Vectors Conditionally Expressing Protein

ABSTRACT

This invention relates to the field of therapeutics. Disclosed are methods of generating conditionally expressing erythropoietin under the control of an ecdysone receptor-based gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals. The methods of the invention cause an in vivo increase in the expression of erythropoietin and an increase in the hematocrit or volume percentage of red blood cells in blood after administration of the ligand.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:SequenceListing.ascii.txt; Size: 153,629 bytes; Date Of Creation: Mar.2, 2012) filed with this application is incorporated herein by referencein its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

Interleukin-12 (IL-12) is a member of the type I cytokine familyinvolved in contributing to a number of biological processes including,but not limited to, protective immune response and suppression oftumorigenesis (Abdi et al., 2006; Adorini, 1999; Adorini, 2001; Adoriniet al., 2002; Adorini et al., 1996; Akhtar et al., 2004; Akiyama et al.,2000; Al-Mohanna et al., 2002; Aliberti et al., 1996; Allavena et al.,1994; Alli and Khar, 2004; Alzona et al., 1996; Amemiya et al., 2006;Araujo et al., 2001; Arulanandam et al., 1999; Athie et al., 2000;Athie-Morales et al., 2004; Bertagnolli et al., 1992; Bhardwaj et al.,1996; Biedermann et al., 2006; Brunda and Gately, 1994; Buchanan et al.,1995; Romani et al., 1997; Rothe et al., 1996; Satoskar et al., 2000;Schopf et al., 1999; Thomas et al., 2000; Tsung et al., 1997; Wolf etal., 1994; Yuminamochi et al., 2007). A growing body of evidencesuggests that IL-12 may be a promising target to control human diseases(e.g., cancer).

Despite the fact that IL-12 remains promising as a cancer therapeuticagent based on its potent supportive activity on Type-1 anti-tumor NKcells, CD4⁺ T cells and CD8⁺ T cells (Trinchieri, 2003), the reportedtoxicity of recombinant human IL-12 (rhIL-12) in patients (Atkins etal., 1997), together with limited sources of GMP-grade rhIL-12 forclinical application, have prevented successful IL-12-based therapeuticapproaches. Thus it seems reasonable that gene therapy approaches mayrepresent safer, more tenable treatment options. Indeed, phase Iclinical trials implementing intra- or peri-tumoral delivery ofrecombinant viral- (Sangro et al., 2004; Triozzi et al., 2005) orplasmid-based IL-12 cDNA (Heinzerling et al., 2005), or IL-12 genemodified autologous fibroblasts (Kang et al., 2001) have been found safeand well-tolerated.

However, objective clinical responses in patients with melanoma or adiverse range of carcinomas receiving these gene therapies have beenrare, variable, transient and largely focused at the site of treatment(Heinzerling et al., 2005; Kang et al., 2001; Sangro et al., 2004;Triozzi et al., 2005). In cases where disease resolution was partial orcomplete, increased frequencies of tumor-infiltrating lymphocytes(Heinzerling et al., 2005; Sangro et al., 2004) and elevated levels ofcirculating tumor-specific CD8⁺ T cells (Heinzerling et al., 2005) havebeen noted, consistent with the improved cross-priming ofantigen-specific T cells in these patients.

Since the cross-priming of specific T cells is best accomplished bydendritic cells (DC) that serve as a natural but regulated source ofIL-12 (Berard et al., 2000), recent reports of the superior pre-clinicalefficacy of DC-based IL-12 gene therapy have been of great interest(Satoh et al., 2002; Tatsumi et al., 2003; Yamanaka et al., 2002). Forexample, it was shown that intratumoral (i.t.) injection of DCengineered to produce IL-12p70 (via recombinant adenovirus infection)results in the dramatically improved cross-priming of abroadly-reactive, tumor-specific CD8⁺ T cell repertoire in concert withtumor rejection in murine models (Tatsumi et al., 2003). Given theprevious use of a recombinant adenovirus encoding mIL-12 under aCMV-based promoter (rAd.cIL12, (Tatsumi et al., 2003)), engineered DCproduction of IL-12 was constitutive, hence the immunologic impact ofthis cytokine early within the tumor lesion and later withintumor-draining lymph nodes could not be resolved with regards totherapeutic outcome. Thus, a need exists for DC engineered forconditional expression of IL-12 for the purpose of regulating both thelevel of transgene expression and the timing of the transgeneactivation. The invention provides a promising therapeutic outcome forthe use of such cells.

In view of the problems associated with gene expression of genes throughvector compositions containing the protein encoded by the nucleic acidsequence of interest in, there remains a need for an improved transfervector compositions to be used for direct injection or for use in cellbased therapies.

Erythropoietin (EPO) plays a central role in the regulation of red bloodcell production by controlling the proliferation, differentiation andsurvival of erythroid progenitors in the bone marrow. Lack of EPOprotein leads to anemia. Treatment with recombinant human EPO (huEPO) isefficient and safe in improving the management of the anemia associatedwith chronic disease. Despite the success of protein therapy variousadverse effects have been reported. For example, patients can become EPOresistant and also hyporesponsive to the biologic. Furthermore,observations in clinical trials with patients that suffer from anemiadue to chemotherapy indicated that rHuEPO protein increases lethality.Thus, there remains a need in the art for an EPO delivery approach fortreating anemia that avoids the disadvantages of presently availabledelivery approaches.

Multiple sclerosis is an inflammatory disease in which the fatty myelinsheaths around the axons of the brain and spinal cord are damaged,leading to demyelination and scarring as well as a broad spectrum ofsigns and symptoms. Although much is known about the mechanisms involvedin the disease process, the cause remains unknown. There is no knowncure for multiple sclerosis. Treatments attempt to return function afteran attack, prevent new attacks, and prevent disability. Multiplesclerosis medications can have adverse effects or be poorly tolerated,and many patients pursue alternative treatments, despite the lack ofsupporting scientific study.

Angioedema is the rapid swelling (edema) of the dermis, subcutaneoustissue, mucosa and submucosal tissues. Angioedema is classified aseither acquired or hereditary. Acquired angioedema is usually caused byallergy and occurs together with other allergic symptoms and urticaria.It can also happen as a side-effect to certain medications, particularlyACE inhibitors. Hereditary angioedema (HAE) exists in three forms, allof which are caused by a genetic mutation that is inherited in anautosomal dominant form. They are distinguished by the underlyinggenetic abnormality. All forms of HAE lead to abnormal activation of thecomplement system, and all forms can cause swelling elsewhere in thebody, such as the digestive tract. If HAE involves the larynx it cancause life-threatening asphyxiation

Pulmonary hypertension is a disorder of the lung in which the pressurein the pulmonary artery (the blood vessel that leads from the heart tothe lungs) rises above normal levels. Pulmonary arterial hypertension(PAH), is a disease characterized by increased pulmonary artery pressureand pulmonary vascular resistance. Harrison's Principles of InternalMedicine, 15th ed., pp. 1506-1507 (McGraw-Hill, 2001). Left untreated,PAH “usually has a dismal prognosis culminating in right ventricularfailure and death.” Ulrich, S., et al., Swiss Med. Wkly 137:73-82, 73(2007).

Crohn's disease is a chronic inflammatory disorder of thegastrointestinal (GI) tract that is defined by relapsing and remittingepisodes, with progression over time to complications of stricture,fistulas, or abscesses. In the U.S. Crohn's disease affectsapproximately one million individuals and the estimated annualdisease-attributable direct costs of IBD (inflammatory bowel disease)have been estimated at $6.3 billion, with Crohn's disease estimated ascontributing $3.6 billion of the costs in that figure. Azathioprine and6-mercaptopurine are frequently prescribed for patients in whomfirst-line therapies fail—in particular, those who are dependent on ordo not have a response to systemic corticosteroids. Approximately 40% ofpatients treated with azathioprine remain in remission at 1 year.Infliximab and other monoclonal antibodies targeting tumor necrosisfactor (TNF) have shown efficacy in inducing and maintaining remissionin patients with Crohn's disease, however in most guidelines andconsensus articles, infliximab is considered the last medical resortbefore handing over the patient to the surgeon in the case of luminaldisease. Hence, there continues to be a need for improved therapeuticmethods for treating IBD and Crohn's disease.

IL-10 is a cytokine produced by activated Th2 cells, B cells,keratinocytes, monocytes, and macrophages. IL-10 inhibits the synthesisof a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF andGM-CSF produced by activated macrophages and by helper T-cells. IL-10 isuseful in promoting growth and differentiation of activated human Bcells, inhibiting Th1 responses to prevent transplant rejection and Tcell-mediated autoimmune diseases.

IL-10 (Interleukin-10) is an immunoregulatory cytokine that stronglydownregulates the production of proinflammatory cytokines, and isinvolved in regulating intestinal inflammation. Clinical development ofrhIL-10 (recombinant human IL-10) demonstrated a narrow therapeuticwindow and a pharmacokinetic profile leading to limited bioavailabilityfor GI tissue. However, systemic adverse effects may not occur if IL-10could be increased locally through in vivo cellular expression.

CF (Cystic Fibrosis) is an autosomal recessive disorder caused bydefects in the gene for the CFTR (cystic fibrosis transmembraneconductance regulator) that result in abnormalities of chloridetransport across epithelial cells on mucosal surfaces, because the CFTRprotein functions as a chloride ion channel across the membrane of cellswhich produce mucus, sweat, saliva, tears, and digestive enzymes. Thetransport of chloride ions helps control the movement of water intissues, which is necessary for the production of thin, freely flowingmucus (normal quantities of which are necessary for protecting thelining of the airways, digestive system, reproductive system, and otherorgans and tissues). The CFTR protein also regulates the function ofother channels, such as those that transport positively chargedparticles called sodium ions across cell membranes. These channels arenecessary for the normal function of organs such as the lungs andpancreas. Thus, CF affects exocrine gland function and causes a buildupof mucus in the lungs, pancreas, and other organs. This mucusobstruction can lead to infection and inflammation of the lungs, inaddition to pancreatic enzyme insufficiency and problems with digestion.

Approximately 30,000 Americans have CF, making it one of the most commonlife-shortening inherited diseases in the United States, with a 37-yearlife expectancy of each CF patient. The most consistent aspect oftherapy in CF has been maintaining quality of life and treating the lungdamage caused by thick mucus and infection. More than 1,000 mutations inthe CFTR gene have been identified in people with cystic fibrosis. Mostof these mutations change a single amino acids in the CFTR protein ordelete a small amount of DNA from the CFTR gene. The most commonmutation, called delta F508, is a deletion of one amino acid at position508 in the CFTR protein. The resulting abnormal channel breaks downshortly after it is made, so it never reaches the cell membrane totransport chloride ions. Disease-causing mutations in the CFTR genealter the production, structure, or stability of the chloride channel.All of these changes prevent the channel from functioning properly,which impairs the transport of chloride ions and the movement of waterinto and out of cells. As a result, cells that line the passageways ofthe lungs, pancreas, and other organs produce mucus that is abnormallythick and sticky. The abnormal mucus obstructs the airways and glands,leading to the characteristic signs and symptoms of cystic fibrosis.Recently KALYDECO™ has been approved by the FDA as the first treatmenttargeting an underlying cause of CF (a G551D mutation in the CFTR gene).However, KALYDECO™ is not effective in treating patients having the mostcommon CFTR mutation. Accordingly, there remains a need for improvedtreatment of CF.

Diabetes mellitus, often simply referred to as diabetes, is a group ofmetabolic diseases in which a person has high blood sugar, eitherbecause the body does not produce enough insulin, or because cells donot respond to the insulin that is produced. This high blood sugarproduces the classical symptoms of polyuria (frequent urination),polydipsia (increased thirst) and polyphagia (increased hunger).Metabolic syndrome is a combination of medical disorders that, whenoccurring together, increase the risk of developing cardiovasculardisease and diabetes. It affects one in five people in the United Statesand prevalence increases with age. Some studies have shown theprevalence in the USA to be an estimated 25% of the population.Accordingly, there remains a need for improved treatment of diabetesmellitus.

Glucagon-like peptide-1 (GLP-1) is a potent antihyperglycemic hormone,inducing glucose-dependent stimulation of insulin secretion whilesuppressing glucagon secretion. GLP-1 appears to restore the glucosesensitivity of pancreatic β-cells, with the mechanism possibly involvingthe increased expression of GLUT2 and glucokinase. GLP-1 is also knownto inhibit pancreatic β-cell apoptosis and stimulate the proliferationand differentiation of insulin-secreting β-cells. In addition, GLP-1inhibits gastric secretion and motility. This delays and protractscarbohydrate absorption and contributes to a satiating effect.

Glucagon-like peptide-2 (GLP-2) is produced by the intestinal endocrineL cell and by various neurons in the central nervous system. IntestinalGLP-2 is co-secreted along with GLP-1 upon nutrient ingestion. Whenexternally administered, GLP-2 produces a number of effects, includingintestinal growth, enhancement of intestinal function, reduction in bonebreakdown and neuroprotection.

Adiponectin is a protein hormone that modulates a number of metabolicprocesses, including glucose regulation and fatty acid catabolism.Adiponectin is exclusively secreted from adipose tissue (and also fromthe placenta in pregnancy) into the bloodstream and is very abundant inplasma relative to many hormones. Levels of the hormone are inverselycorrelated with body fat percentage in adults.

Human leptin is manufactured primarily in the adipocytes of whiteadipose tissue, and the level of circulating leptin is directlyproportional to the total amount of fat in the body. Leptin acts onreceptors in the hypothalamus of the brain where it inhibits appetite by(1) counteracting the effects of neuropeptide Y (a potent feedingstimulant secreted by cells in the gut and in the hypothalamus); (2)counteracting the effects of anandamide (another potent feedingstimulant that binds to the same receptors as THC), and (3) promotingthe synthesis of α-MSH, an appetite suppressant.

SUMMARY OF THE INVENTION

The present invention provides a recombinant vector encoding protein(s)having the function(s) of one or more therapeutic proteins (e.g.,immunomodulators), under the control of one or more promoters. In oneembodiment, the one or more promoters are conditional. In anotherembodiment, the one or more promoters are constitutive. In anotherembodiment, the vector is an adenovirus vector encoding the protein(s)driven off a promoter that can be conditionally activated by provisionof a soluble small molecule ligand such as diacylhydrazines (e.g.,RG-115819, RG-115830 or RG-115932).

The present invention also provides a method of inducing, regulating, orenhancing erythropoietin (EPO) expression in a mammal, wherein themethod comprises:

(a) administering intramuscularly to the mammal an adeno-associatedvirus wherein the virus comprises a polynucleotide encoding EPO; and

(b) administering an activator ligand,

wherein the adeno-associated virus further comprises a gene switch,wherein the gene switch comprises at least one transcription factorsequence operably linked to a promoter,

wherein at least one transcription factor encoded by the at least onetranscription factor sequence is a ligand-dependent transcriptionfactor,

wherein the adeno-associated virus further comprises a second promoteroperably linked to the polynucleotide encoding EPO, and wherein thesecond promoter is activated by the at least one ligand-dependenttranscription factor following administration of activator ligand.

The present invention also provides a vector comprising a polynucleotideencoding a gene switch, wherein the polynucleotide comprises (1) atleast one transcription factor sequence which is operably linked to apromoter, wherein the at least one transcription factor sequence encodesa ligand-dependent transcription factor, and (2) a polynucleotideencoding one or more proteins operably linked to a promoter which isactivated by the ligand-dependent transcription factor, wherein the oneor more proteins is selected from the group consisting of a C1 esteraseinhibitor, a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

The present invention also provides a method of producing a populationof cells expressing one or more proteins, wherein the method comprisesmodifying the cells with a recombinant vector conditionally expressingone or more proteins, wherein the vector comprises a polynucleotideencoding a gene switch, wherein the polynucleotide comprises (1) atleast one transcription factor sequence operably linked to a promoter,wherein the at least one transcription factor sequence encodes aligand-dependent transcription factor, and (2) a polynucleotide encodingone or more proteins linked to a promoter which is activated by theligand-dependent transcription factor, wherein the one or more proteinsare selected from the group consisting of a C1 esterase inhibitor, akallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

The present invention also provides a population of cells which havebeen modified with a recombinant vector conditionally expressing one ormore proteins, wherein the vector comprises a polynucleotide encoding agene switch, wherein the polynucleotide comprises (1) at least onetranscription factor sequence operably linked to a promoter, wherein theat least one transcription factor sequence encodes a ligand-dependenttranscription factor, and (2) a polynucleotide encoding one or moreproteins selected from the group consisting of a C1 esterase inhibitor,a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

The present invention also provides an in vitro engineered cellcomprising a recombinant polynucleotide encoding a gene switch, whereinthe polynucleotide encoding a gene switch comprises (1) at least onetranscription factor sequence, wherein the at least one transcriptionfactor sequence encodes a ligand-dependent transcription factor,operably linked to a promoter, and (2) a polynucleotide encoding one ormore proteins linked to a promoter which is activated by theligand-dependent transcription factor, wherein the one or more proteinsis selected from the group consisting of a C1 esterase inhibitor, akallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

The present invention also provides a method for treating a disease in amammal, comprising:

(a) administering a population of cells in vitro engineered toconditionally express one or more proteins; and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands;

thereby inducing expression of the one or more proteins, wherein the oneor more proteins is selected from the group consisting of a C1 esteraseinhibitor, a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

The present invention also provides a method for treating a disease in amammal, comprising:

(a) administering to the mammal a vector for conditionally expressingone or more proteins, the vector comprising a polynucleotide encoding agene switch, wherein the polynucleotide comprises

(1) at least one transcription factor sequence which is operably linkedto a promoter, wherein the at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and

(2) a polynucleotide encoding one or more proteins operably linked to apromoter which is activated by the ligand-dependent transcriptionfactor,

wherein the vector is not contained within a cell; and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands; thereby inducing expression of the oneor more proteins and treating the disease,

wherein the one or more proteins is selected from the group consistingof a C1 esterase inhibitor, a kallikrein inhibitor, a bradykinin B2receptor inhibitor, a prostaglandin synthase, a glucagon-like peptide-1(GLP-1), a glucagon-like peptide-2 (GLP-2), adiponectin, leptin, andcystic fibrosis transmembrane conductance regulator (CFTR).

The present invention also provides a method for treating multiplesclerosis in a mammal, comprising:

(a) administering to the mammal a vector for conditionally expressingone or more proteins, the vector comprising a polynucleotide encoding agene switch, wherein the polynucleotide comprises

(1) at least one transcription factor sequence which is operably linkedto a promoter, wherein the at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and

(2) a polynucleotide encoding one or more proteins operably linked to apromoter which is activated by the ligand-dependent transcriptionfactor,

wherein the vector is not contained within a cell; and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands; thereby inducing expression of the oneor more proteins and treating the disease,

wherein the one or more proteins is selected from the group consistingof myelin basic protein (MBP) and interferon-beta (IFN-B).

A method for treating inflammatory bowel or Crohn's disease in a mammal,comprising:

(a) administering to the mammal a vector for conditionally expressingone or more proteins, the vector comprising a polynucleotide encoding agene switch, wherein the polynucleotide comprises

(1) at least one transcription factor sequence which is operably linkedto a promoter, wherein the at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and

(2) a polynucleotide encoding one or more proteins operably linked to apromoter which is activated by the ligand-dependent transcriptionfactor, and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands; thereby inducing expression of the oneor more proteins and treating the disease,

wherein one of the one or more proteins is interleukin-10 (IL-10).

DETAILED DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are line graphs that depict the potency of Ad-RTS-mIL12with activator ligand in contralateral melanoma (B16F0) tumor in C57Bl6mice. FIG. 1A depicts the volume of the treated tumor on the rightflanks of the animals. FIG. 1B depicts the volume of the untreated tumoron the left flanks of the animals. Tumor sizes are shown as mean±SE.

FIG. 2 is a line graph that depicts changes in body weight of micebearing melanoma tumors on both flanks, in response to Ad-RTS-mIL 12 andactivator ligand.

FIG. 3 is a bar graph that depicts the in vitro regulated expression ofmIL12 and mIFNα in the LLC and 4T1 cell lines.

FIGS. 4A and 4B are line graphs that depict the effect of mIL12 andmIFNα in the LLC and 4T1 cell lines.

FIG. 5 is a bar graph that depicts the systemic and intratumoral effectsof IL12 and IFNα in mice.

FIG. 6 is a combined line and bar graph that depicts that theco-expression of IL12 and IFNα enhances MHC Class I expression in 4T1and LLC cancer cells.

FIGS. 7A, 7B, 7C and 7D depict vector maps for vectors 0034A, 0034B,0034CB and 0034D, respectively. 0034A carries standard switch systemelements. 0034B carries a modified regulated promoter.

FIG. 8 is a line graph that depicts the physiological response of C3H/Hmice following intramuscular (IM) administration of AAV-HuEPO.

FIG. 9 is a combined line graph and bar graph that shows the effect ofregulated expression of HuEPO on hematocrit in C3H/H mice.

FIG. 10 is a combined line graph and bar graph that shows the effect ofregulated expression of HuEPO on hematocrit in Balb/c mice.

FIG. 11 is a combined line graph and bar graph that shows the effect ofregulated expression of HuEPO on hematocrit following a single IMinjection of AAV-HuEPO.

FIGS. 12A and 12B are line graphs that show the absolute changes inhematocrit following IM delivery of AAV-HuEPO.

FIG. 13 is a combined line graph and bar graph that shows the effect ofregulated expression of HuEPO on hematocrit as a function of activatorligand dose.

FIG. 14 is a combined line graph and bar graph that shows the effect ofthe expression of HuEPO in 3/4 nephrectomized C3H/H mice results onhematocrit.

FIG. 15 is a bar graph that shows the effect of the expression of IMdelivery of RTS-HuEPO to Balb/c mice on hematocrit.

DESCRIPTION OF SEQUENCE LISTING

SEQ ID NO: 1 is an amino acid sequence of an ecdysone response elementfound in Drosophila.

SEQ ID NO: 2 is a nucleic acid sequence of an ecdysone response elementfound in Drosophila melanogaster.

SEQ ID NO: 3 is a nucleic acid sequence of an ecdysone response elementfound in Drosophila melanogaster.

SEQ ID NO: 4 is a DNA sequence of adenovirus vector comprising humanIL-12 coding sequence: Ad-RTS-hIL-12 (SP1-RheoIL-12).

SEQ ID NO: 5 is a nucleic acid sequence for the vector (Ad-RTS-mIL-12).

SEQ ID NO: 6 is the amino acid sequence for human erythropoietin.

SEQ ID NO: 7 is the amino acid sequence for the Choristoneura fumiferanaecdysone receptor ligand binding domain.

SEQ ID NO: 8 is the nucleic acid sequence for the signal peptidesequence, human erythropoietin sequence, and stop codon.

SEQ ID NO: 9 is the nucleic acid sequence for human myelin basicprotein.

SEQ ID NO: 10 is the amino acid sequence for human C1 esteraseinhibitor.

SEQ ID NO: 11 is the amino acid sequence for ecallantide.

SEQ ID NO: 12 is the amino acid sequence for human prostaglandinsynthetase 2.

SEQ ID NO: 13 is the amino acid sequence for human prostaglandinsynthetase 1.

SEQ ID NO: 14 is the nucleic acid sequence for human prostaglandinsynthetase 2.

SEQ ID NO: 15 is the nucleic acid sequence for human prostaglandinsynthetase 1

SEQ ID NO: 16 is the amino acid sequence for human interferon-beta.

SEQ ID NO: 17 is the amino acid sequence for human GLP-1.

SEQ ID NO: 18 is the amino acid sequence for human GLP-2.

SEQ ID NO: 19 is the amino acid sequence for human adiponectin.

SEQ ID NO: 20 is the amino acid sequence for human leptin.

SEQ ID NO: 21 is the amino acid sequence for human CFTR.

SEQ ID NO: 22 is the amino acid sequence for human IL-10.

DETAILED DESCRIPTION OF INVENTION Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference andunderstanding, and the inclusion of such definitions herein should notnecessarily be construed to mean a substantial difference over what isgenerally understood in the art. Commonly understood definitions ofmolecular biology terms and/or methods and/or protocols can be found inRieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991; Lewin, Genes V, OxfordUniversity Press: New York, 1994; Sambrook et al., Molecular Cloning, ALaboratory Manual (3d ed. 2001) and Ausubel et al., Current Protocols inMolecular Biology (1994). As appropriate, procedures involving the useof commercially available kits and/or reagents are generally carried outin accordance with manufacturer's guidance and/or protocols and/orparameters unless otherwise noted.

The invention provides a recombinant vector encoding protein(s), underthe control of one or more promoters. In one embodiment, the one or morepromoters are conditional. In another embodiment, the one or morepromoters are constitutive. In another embodiment, the vector is anadenovirus vector encoding the protein(s) driven off a promoter that canbe conditionally activated by provision of a soluble small moleculeligand such as diacylhydrazines (e.g., RG-115819, RG-115830 orRG-115932). This vector allows for the control of expression of theprotein(s) in cells.

In one embodiment, the polynucleotide coding for the one or moreproteins having the functions of the immunomodulator is under control ofthe promoter of the gene switch and the polynucleotide coding for aprotein having the function of IL-12 is under control of a constitutivepromoter. In another embodiment, both the polynucleotide coding forprotein(s) having the functions of the therapeutic proteins (e.g.,immunomodulators) and the polynucleotide coding for a protein having thefunction of IL-12 are both under control of a multicistronic promoter ofthe gene switch. In another embodiment, the polynucleotide coding for aprotein(s) having the function of the therapeutic proteins (e.g.,immunomodulators) is under control of the promoter of the gene switchand the polynucleotide coding for a protein having the function of IL-12is under control of a conditional promoter which is different than thegene switch promoter. In a further embodiment, the gene regulationsystem for the polynucleotide coding for the protein(s) having thefunction of the therapeutic proteins (e.g., immunomodulators) and thegene regulation system for the polynucleotide having the function ofIL-12 are orthogonal. In a further embodiment, the gene regulationsystem for each polynucleotide coding for each protein is orthogonal.

In one embodiment, the invention also provides a treatment of cancer,such as, but not limited to, melanoma tumors, glioma tumors, renalcancer, and prostate cancers, as well as the cancers listed herein inTable 1. IL-12 gene therapy has demonstrated anti-tumor efficacy inanimal model studies when applied as a recombinant cDNA vector (Faure etal., 1998; Sangro et al., 2005), but even more so, when applied in thecontext of gene-modified DC (Satoh et al., 2002; Svane et al., 1999;Tatsumi et al., 2003; Yamanaka et al., 2002). To date, however, humanphase I trials of IL-12 gene therapy implementing plasmids or viralvectors have failed to achieve durable, objective clinical responses inthe cancer setting (Heinzerling et al., 2005; Kang et al., 2001; Sangroet al., 2004; Triozzi et al., 2005) gene therapy as described hereinprovides a promising therapeutic modality.

In one embodiment, the invention provides a method for treating a tumorin a mammal, comprising the steps of:

(a) administering intratumorally to tumor microenvironments, in the areasurrounding the tumor, or systemically a population of immune cells,TSCs or vectors of the invention (or a combination thereof), which arein vitro engineered to conditionally express one or more proteins havingthe function of a therapeutic protein (e.g., immunomodulator); and

(b) administering to said mammal a therapeutically effective amount ofan activating ligand;

thereby inducing expression of a protein having the function of thetherapeutic protein (e.g., immunomodulator) and treating said tumor.

In another embodiment, the invention provides a method for treating adisease or disorder in a mammal, comprising the steps of:

(a) administering to said mammal a population of modified cells, whichare modified to conditionally express one or more proteins having thefunction of an therapeutic protein (e.g., immunomodulator); and

(b) administering to said mammal a therapeutically effective amount ofan activating ligand;

thereby inducing expression of a protein having the function of thetherapeutic protein (e.g., immunomodulator) and treating said disease ordisorder.

In another embodiment, the invention provides a method for treating adisease or disorder in a mammal, comprising the steps of:

(a) administering to said mammal two or more populations of modifiedcells, which are modified to conditionally express one or more proteinshaving the function of a therapeutic protein (e.g., immunomodulator),wherein each population of modified cells expresses a different set ofone or more therapeutic proteins (e.g., immunomodulators); and

(b) administering to said mammal a therapeutically effective amount ofone or more activating ligands;

thereby inducing expression of proteins having the function of thetherapeutic proteins (e.g., immunomodulators) and treating said diseaseor disorder.

In another embodiment, the invention provides a method for treating adisease or disorder in a mammal, comprising the steps of:

(a) administering to said mammal a population of a modified cells, whichare modified to conditionally express one or more proteins having thefunction of a therapeutic protein (e.g., immunomodulator) and a proteinhaving the function of IL-12, wherein at least one of the proteinshaving the function of the therapeutic protein (e.g., immunomodulator)or IL-12 is under control of a conditional promoter that is activated bya ligand; and

(b) administering to said mammal a therapeutically effective amount ofthe activating ligand;

thereby inducing expression of a protein having the function of thetherapeutic protein (e.g., immunomodulator) and/or the protein havingthe function of IL-12 and treating said disease or disorder.

In another embodiment, the invention provides a method for treating adisease or disorder in a mammal, comprising the steps of:

(a) administering to said mammal two or more populations of modifiedcells, which are modified to conditionally express one or more proteinshaving the function of a therapeutic protein (e.g., immunomodulator) anda protein having the function of IL-12, wherein each population ofmodified cells expresses a different set of one or more proteins havingthe function of a therapeutic protein (e.g., immunomodulator), whereinat least one of the proteins having the function of the therapeuticprotein (e.g., immunomodulator) or IL-12 is under control of aconditional promoter that is activated by a ligand; and

(b) administering to said mammal a therapeutically effective amount ofone or more activating ligands;

thereby inducing expression of a protein having the function of thetherapeutic proteins (e.g., immunomodulators) and/or the protein havingthe function of IL-12 and treating said disease or disorder.

In one embodiment, the invention provides a vector for conditionallyexpressing protein(s) comprising a polynucleotide encoding a geneswitch, wherein said polynucleotide encoding a gene switch comprises (1)at least one transcription factor sequence operably linked to apromoter, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and (2) apolynucleotide encoding one or more proteins linked to a promoter whichis activated by said ligand-dependent transcription factor.

In one embodiment, the vector of the invention conditionally expressesthe protein. In another embodiment, the vector, e.g., adenoviral vector,conditionally expressing one or more proteins, further comprises anucleic acid sequence encoding a signal peptide. The signal peptide canbe codon-optimized. In other embodiments, the vector further comprises5′ untranslated region (UTR), 3′ regulatory region, or both and improvesprotein expression and/or overall yield.

The invention further provides a method of producing a population ofcells, expressing protein(s), by modifying (e.g., transfecting,electroporating, etc.) the cells with a recombinant vector conditionallyexpressing protein(s), wherein the vector comprises a polynucleotideencoding a gene switch, wherein said polynucleotide comprises (1) atleast one transcription factor sequence operably linked to a promoter,wherein said at least one transcription factor sequence encodes aligand-dependent transcription factor, and (2) a polynucleotide encodingone or more proteins linked to a promoter which is activated by saidligand-dependent transcription factor.

In some embodiments, the invention provides a method of increasingexpression of a protein comprising generating the vector conditionallyexpressing one or more proteins and one or more regulatory sequence,wherein said one or more regulatory sequence improves expression of theprotein.

The invention further provides a population of cells expressingprotein(s), which has been modified (e.g., transfected, electroporated,etc.) with a recombinant vector conditionally the expressing protein(s),wherein the vector comprises a polynucleotide encoding a gene switch,wherein said polynucleotide comprises (1) at least one transcriptionfactor sequence operably linked to a promoter, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, and (2) a polynucleotide encoding one or more proteins linked tothe promoter which is activated by said ligand-dependent transcriptionfactor.

In another embodiment, the invention provides a composition comprisingtwo or more populations of cells of the present invention, wherein eachpopulation of cells in the composition expresses one or more proteinsthat are different from the one or more proteins expressed in the otherpopulation(s) of cells in the composition. In one embodiment, thecomposition contains two populations of cells. In another embodiment,the composition contains more than two populations of cells. In anotherembodiment, the composition contains three populations of cells. Inanother embodiment, the composition contains four populations of cells.

In another embodiment, the invention provides an in vitro engineeredcell comprising a vector comprising a polynucleotide encoding a geneswitch, wherein said polynucleotide comprises (1) at least onetranscription factor sequence operably linked to a promoter, whereinsaid at least one transcription factor sequence encodes aligand-dependent transcription factor, and (2) a polynucleotide encodinga protein linked to a promoter which is activated by saidligand-dependent transcription factor.

In another embodiment, the invention provides a composition comprisingtwo or more populations of in vitro engineered cells, wherein each ofthe populations of in vitro engineered cells in the compositioncomprises a vector comprising a polynucleotide encoding a gene switch,wherein said polynucleotide comprises (1) at least one transcriptionfactor sequence operably linked to a promoter, wherein said at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, and (2) a polynucleotide encoding a protein linked to a promoterwhich is activated by said ligand-dependent transcription factor, andwherein each population of in vitro engineered cells in the compositionexpresses one or more proteins that are different from the one or moreproteins expressed in the other population(s) of in vitro engineeredcell in the composition.

In another embodiment, vectors and methods of the present invention areused to treat diabetes mellitus, metabolic disease, metabolic disorderand metabolic syndrome. In one embodiment, the vector comprises apolynucleotide sequence encoding GLP-1, GLP-2, adiponectin or leptin, ora fragment thereof of GLP-1, GLP-2, adiponectin or leptin. In anotherembodiment, the vector comprises a polynucleotide sequence encodinghuman GLP-1, GLP-2, adiponectin or leptin, or a fragment thereof ofhuman GLP-1, GLP-2, adiponectin or leptin.

In another embodiment, vectors and methods of the present invention areused to treat inflammatory bowel disease (IBD) and/or Crohn's disease.In one embodiment, the vector comprises a polynucleotide sequenceencoding IL-10, or a fragment thereof. In another embodiment, the vectorcomprises a polynucleotide sequence encoding human IL-10, or a fragmentthereof.

In one embodiment, the present invention encompasses a vector or vectorscomprising constitutive, inducible, or tissue-specific promoters whichallow for in vivo production of the cystic fibrosis transmembraneconductance regulator (CFTR) to replace or supplement the deficient ormutant CFTR protein. Thus, in one embodiment, vectors and methods of thepresent invention are used to treat CF (Cystic Fibrosis). In oneembodiment, the vector comprises a polynucleotide sequence encoding anormal (non-mutatant) CFTR, or a functional (bioactive/ion transportcapable) fragment thereof. In another embodiment, the vector comprises apolynucleotide sequence encoding a normal human CFTR (non-mutatantCFTR), or a functional (bioactive/ion transport capable) fragmentthereof.

The invention also provides a pharmaceutical composition comprising apopulation of cells, as described herein or a composition suitable fordirect injection of the expression vectors absent a population of cells,i.e., directly injected.

The present invention also provides a vector for conditionallyexpressing a prostaglandin synthase, the vector comprising apolynucleotide encoding a gene switch, wherein the polynucleotidecomprises (1) at least one transcription factor sequence which isoperably linked to a promoter, wherein the at least one transcriptionfactor sequence encodes a ligand-dependent transcription factor, and (2)a polynucleotide encoding a prostaglandin synthase. The presentinvention also provides a method for treating pulmonary hypertensioncomprising administering the vector.

IL-12 is a cytokine that can act as a growth factor for activated T andNK cells, enhance the lytic activity of NK/lymphokine-activated Killercells, and stimulate the production of IFN-gamma by resting peripheralblood mononuclear cells (PBMC). The polynucleotide sequences of IL-12are available from public databases as accession numbers NM_000882(human IL12A); NM_002187 (human IL12B); NM_008351 (mouse IL12a);NM_008352 (mouse IL12b); NM_213588 (chicken IL12A); NM_213571 (chickenIL12B); NM_053390 (rat IL12a); and NM_022611 (rat IL12b), sequences ofwhich are incorporated by reference herein.

The amino acid sequences of interleukin 12 (IL-12) are available frompublic databases as accession numbers NP_000873 (human IL12A); NP_002178(human IL12B); NP_032377 (mouse IL12a); NP_032378 (mouse 112b);NP_998753 (chicken IL12A); NP_998736 (chicken IL12B); NP_445842 (ratIL12a); and NP_072133 (rat IL12b), sequences of which are incorporatedby reference herein.

In one embodiment, the IL-12 gene is the wild type mouse IL-12 sequence.In another embodiment, the sequence is at least 85% identical to wildtype mouse IL-12, e.g., at least 90%, 95%, or 99% identical to wild typemouse IL-12. In a further embodiment, the IL-12 gene sequence encodesthe mouse IL-12 polypeptide. In another embodiment, the gene encodes apolypeptide that is at least 85% identical to wild type mouse IL-12,e.g., at least 90%, 95%, or 99% identical to wild type mouse IL-12.

Myelin basic protein (MBP) is a protein believed to be important in theprocess of myelination of nerves in the central nervous system. Theprotein encoded by the classic MBP gene is a major constituent of themyelin sheath of oligodendrocytes and Schwann cells in the nervoussystem. MBP-related transcripts are also present in the bone marrow andthe immune system. Amino acid and polynucleotide sequences for MBP areavailable as accession numbers NM_001025081 and NP_001020252 (human) andNM_001025245 and NP_001020416 (mouse).

In one embodiment, the MBP is the wild type human MBP sequence. Inanother embodiment, the sequence is at least 85% identical to wild typehuman MBP, e.g., at least 90%, 95%, or 99% identical to wild type humanMBP.

C1 esterase inhibitor (C1-inhibitor, C1-inh) is a protease inhibitorbelonging to the serpin superfamily. Its main function is the inhibitionof the complement system to prevent spontaneous activation. C1 esteraseinhibitor is an acute-phase protein that circulates in blood at levelsof around 0.25 g/L, and its level rises approximately 2-fold duringinflammation. C1 esterase inhibitor irreversibly binds to andinactivates C1r and C1s proteases in the C1 complex of classical pathwayof complement. MASP-1 and MASP-2 proteases in MBL complexes of thelectin pathway are also inactivated. C1 esterase inhibitor prevents theproteolytic cleavage of later complement components C4 and C2 by C1 andMBL. C1 esterase inhibitor also inhibits proteases of the fibrinolytic,clotting, and kinin pathways. C1 esterase inhibitor is an inhibitor ofplasma kallikrein. The amino acid sequence for human C1 esteraseinhibitor is found at GenBank ADU87625.1, Accession GU727623.1).

In one embodiment, the C1 esterase inhibitor is the wild type human C1esterase inhibitor sequence. In another embodiment, the sequence is atleast 85% identical to wild type human MBP, e.g., at least 90%, 95%, or99% identical to wild type human C1 esterase inhibitor.

Ecallantide (trade name Kalbitor, investigational name DX-88) is aninhibitor of the protein kallikrein, and is useful in the treatment ofhereditary angioedema (HAE) and in the prevention of blood loss incardiothoracic surgery. The amino acid sequence for ecallantide is foundin U.S. Patent Publication NO. 2007/0213275, which is incorporated byreference in its entirety.

In one embodiment, the ecallantide is the wild type human ecallantidesequence. In another embodiment, the sequence is at least 85% identicalto wild type human MBP, e.g., at least 90%, 95%, or 99% identical towild type human ecallantide.

In one embodiment, the GLP-1 is the wild type human GLP-1 sequence. Inanother embodiment, the sequence is at least 85% identical to wild typehuman GLP-1, e.g., at least 90%, 95%, or 99% identical to wild typehuman GLP-1.

In one embodiment, the GLP-2 is the wild type human GLP-2 sequence. Inanother embodiment, the sequence is at least 85% identical to wild typehuman GLP-2, e.g., at least 90%, 95%, or 99% identical to wild typehuman GLP-2.

In one embodiment, the adiponectin is the wild type human adiponectinsequence. In another embodiment, the sequence is at least 85% identicalto wild type human adiponectin, e.g., at least 90%, 95%, or 99%identical to wild type human adiponectin.

In one embodiment, the leptin is the wild type human leptin sequence. Inanother embodiment, the sequence is at least 85% identical to wild typehuman leptin, e.g., at least 90%, 95%, or 99% identical to wild typehuman leptin.

In one embodiment, the IL-10 is the wild type human IL-10 sequence. Inanother embodiment, the sequence is at least 85% identical to wild typehuman IL-10, e.g., at least 90%, 95%, or 99% identical to wild typehuman IL-10.

The term “isolated” for the purposes of the invention designates abiological material (cell, nucleic acid or protein) that has beenremoved from its original environment (the environment in which it isnaturally present). For example, a polynucleotide present in the naturalstate in a plant or an animal is not isolated, however the samepolynucleotide separated from the adjacent nucleic acids in which it isnaturally present, is considered “isolated.”

The term “purified,” as applied to biological materials does not requirethe material to be present in a form exhibiting absolute purity,exclusive of the presence of other compounds. It is rather a relativedefinition.

“Nucleic acid,” “nucleic acid molecule,” “oligonucleotide,”“nucleotide,” and “polynucleotide” are used interchangeably and refer tothe phosphate ester polymeric form of ribonucleosides (adenosine,guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNAmolecules”), or any phosphoester analogs thereof, such asphosphorothioates and thioesters, in either single stranded form, or adouble-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, inter alia, inlinear or circular DNA molecules (e.g., restriction fragments),plasmids, supercoiled DNA and chromosomes. In discussing the structureof particular double-stranded DNA molecules, sequences may be describedherein according to the normal convention of giving only the sequence inthe 5′ to 3′ direction along the non-transcribed strand of DNA (i.e.,the strand having a sequence homologous to the mRNA). A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation. DNA includes, but is not limited to, cDNA, genomic DNA,plasmid DNA, synthetic DNA, and semi-synthetic DNA.

The term “fragment,” as applied to polynucleotide sequences, refers to anucleotide sequence of reduced length relative to the reference nucleicacid and comprising, over the common portion, a nucleotide sequenceidentical to the reference nucleic acid. Such a nucleic acid fragmentaccording to the invention may be, where appropriate, included in alarger polynucleotide of which it is a constituent. Such fragmentscomprise, or alternatively consist of, oligonucleotides ranging inlength from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25,30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90,100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000,3000, 4000, 5000, or more consecutive nucleotides of a nucleic acidaccording to the invention.

As used herein, an “isolated nucleic acid fragment” refers to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “gene” refers to a polynucleotide comprising nucleotides that encode afunctional molecule, including functional molecules produced bytranscription only (e.g., a bioactive RNA species) or by transcriptionand translation (e.g., a polypeptide). The term “gene” encompasses cDNAand genomic DNA nucleic acids. “Gene” also refers to a nucleic acidfragment that expresses a specific RNA, protein or polypeptide,including regulatory sequences preceding (5′ non-coding sequences) andfollowing (3′ non-coding sequences) the coding sequence. “Native gene”refers to a gene as found in nature with its own regulatory sequences.“Chimeric gene” refers to any gene that is not a native gene, comprisingregulatory and/or coding sequences that are not found together innature. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A chimericgene may comprise coding sequences derived from different sources and/orregulatory sequences derived from different sources. “Endogenous gene”refers to a native gene in its natural location in the genome of anorganism. A “foreign” gene or “heterologous” gene refers to a gene notnormally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. For example, the interleukin-12 (IL-12) gene encodes theIL-12 protein. IL-12 is a heterodimer of a 35-kD subunit (p35) and a40-kD subunit (p40) linked through a disulfide linkage to make fullyfunctional IL-12p70. The IL-12 gene encodes both the p35 and p40subunits.

“Heterologous DNA” refers to DNA not naturally located in the cell, orin a chromosomal site of the cell. The heterologous DNA may include agene foreign to the cell.

The term “genome” includes chromosomal as well as mitochondrial,chloroplast and viral DNA or RNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook et al. in Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor (1989), particularly Chapter 11 and Table 11.1 therein). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization.

Stringency conditions can be adjusted to screen for moderately similarfragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) of 55°, can be used, e.g., 5×SSC,0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5%SDS. Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SSC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SSC.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The term “complementary” is usedto describe the relationship between nucleotide bases that are capableof hybridizing to one another. For example, with respect to DNA,adenosine is complementary to thymine and cytosine is complementary toguanine. Accordingly, the invention also includes isolated nucleic acidfragments that are complementary to the complete sequences as disclosedor used herein as well as those substantially similar nucleic acidsequences.

In one embodiment of the invention, polynucleotides are detected byemploying hybridization conditions comprising a hybridization step atT_(m) of 55° C., and utilizing conditions as set forth above. In otherembodiments, the T_(m) is 60° C., 63° C., or 65° C.

Post-hybridization washes also determine stringency conditions. One setof conditions uses a series of washes starting with 6×SSC, 0.5% SDS atroom temperature for 15 minutes (min), then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. One set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS isincreased to 60° C. Another set of highly stringent conditions uses twofinal washes in 0.1×SSC, 0.1% SDS at 65° C.

Moreover, the skilled artisan recognizes that substantially similarsequences encompassed by this invention are also defined by theirability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65°C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), withthe sequences exemplified herein. Substantially similar nucleic acidfragments of the invention are those nucleic acid fragments whose DNAsequences are at least about 70%, 80%, 90% or 95% identical to the DNAsequence of the nucleic acid fragments reported herein.

The appropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of T_(m) forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher T_(m)) of nucleic acid hybridizations decreasesin the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids ofgreater than 100 nucleotides in length, equations for calculating T_(m)have been derived (see Sambrook et al., supra, 9.50-0.51). Forhybridization with shorter nucleic acids, i.e., oligonucleotides, theposition of mismatches becomes more important, and the length of theoligonucleotide determines its specificity (see Sambrook et al., supra,11.7-11.8).

In one embodiment of the invention, polynucleotides are detected byemploying hybridization conditions comprising a hybridization step inless than 500 mM salt and at least 37° C., and a washing step in 2×SSPEat a temperature of at least 63° C. In another embodiment, thehybridization conditions comprise less than 200 mM salt and at least 37°C. for the hybridization step. In a further embodiment, thehybridization conditions comprise 2×SSPE and 63° C. for both thehybridization and washing steps.

In another embodiment, the length for a hybridizable nucleic acid is atleast about 10 nucleotides. Preferably a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; e.g., atleast about 20 nucleotides; e.g., at least 30 nucleotides. Furthermore,the skilled artisan will recognize that the temperature and washsolution salt concentration may be adjusted as necessary according tofactors such as length of the probe.

The term “probe” refers to a single-stranded nucleic acid molecule thatcan base pair with a complementary single stranded target nucleic acidto form a double-stranded molecule.

As used herein, the term “oligonucleotide” refers to a short nucleicacid that is hybridizable to a genomic DNA molecule, a cDNA molecule, aplasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g.,with ³²P-nucleotides or nucleotides to which a label, such as biotin,has been covalently conjugated. A labeled oligonucleotide can be used asa probe to detect the presence of a nucleic acid. Oligonucleotides (oneor both of which may be labeled) can be used as PCR primers, either forcloning full length or a fragment of a nucleic acid, for DNA sequencing,or to detect the presence of a nucleic acid. An oligonucleotide can alsobe used to form a triple helix with a DNA molecule. Generally,oligonucleotides are prepared synthetically, preferably on a nucleicacid synthesizer. Accordingly, oligonucleotides can be prepared withnon-naturally occurring phosphoester analog bonds, such as thioesterbonds, etc.

A “primer” refers to an oligonucleotide that hybridizes to a targetnucleic acid sequence to create a double stranded nucleic acid regionthat can serve as an initiation point for DNA synthesis under suitableconditions. Such primers may be used in a polymerase chain reaction orfor DNA sequencing.

“Polymerase chain reaction” is abbreviated PCR and refers to an in vitromethod for enzymatically amplifying specific nucleic acid sequences. PCRinvolves a repetitive series of temperature cycles with each cyclecomprising three stages: denaturation of the template nucleic acid toseparate the strands of the target molecule, annealing a single strandedPCR oligonucleotide primer to the template nucleic acid, and extensionof the annealed primer(s) by DNA polymerase. PCR provides a means todetect the presence of the target molecule and, under quantitative orsemi-quantitative conditions, to determine the relative amount of thattarget molecule within the starting pool of nucleic acids.

“Reverse transcription-polymerase chain reaction” is abbreviated RT-PCRand refers to an in vitro method for enzymatically producing a targetcDNA molecule or molecules from an RNA molecule or molecules, followedby enzymatic amplification of a specific nucleic acid sequence orsequences within the target cDNA molecule or molecules as describedabove. RT-PCR also provides a means to detect the presence of the targetmolecule and, under quantitative or semi-quantitative conditions, todetermine the relative amount of that target molecule within thestarting pool of nucleic acids.

A DNA “coding sequence” or “coding region” refers to a double-strandedDNA sequence that encodes a polypeptide and can be transcribed andtranslated into a polypeptide in a cell, ex vivo, in vitro or in vivowhen placed under the control of suitable regulatory sequences.“Suitable regulatory sequences” refers to nucleotide sequences locatedupstream (5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites and stem-loop structures. Theboundaries of the coding sequence are determined by a start codon at the5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl)terminus. A coding sequence can include, but is not limited to,prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and evensynthetic DNA sequences. If the coding sequence is intended forexpression in an eukaryotic cell, a polyadenylation signal andtranscription termination sequence will usually be located 3′ to thecoding sequence.

“Open reading frame” is abbreviated ORF and refers to a length ofnucleic acid sequence, either DNA, cDNA or RNA, that comprises atranslation start signal or initiation codon, such as an ATG or AUG, anda termination codon and can be potentially translated into a polypeptidesequence.

The term “head-to-head” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-head orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 5′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds away from the 5′ end ofthe other polynucleotide. The term “head-to-head” may be abbreviated(5′)-to-(5′) and may also be indicated by the symbols (← →) or(3′←5′5′→3′).

The term “tail-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a tail-to-tail orientation when the 3′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds toward the otherpolynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′)and may also be indicated by the symbols (→ ←) or (5′→3′3′←5′).

The term “head-to-tail” is used herein to describe the orientation oftwo polynucleotide sequences in relation to each other. Twopolynucleotides are positioned in a head-to-tail orientation when the 5′end of the coding strand of one polynucleotide is adjacent to the 3′ endof the coding strand of the other polynucleotide, whereby the directionof transcription of each polynucleotide proceeds in the same directionas that of the other polynucleotide. The term “head-to-tail” may beabbreviated (5′)-to-(3′) and may also be indicated by the symbols (→ →)or (5′→3′5′→3′).

The term “downstream” refers to a nucleotide sequence that is located 3′to a reference nucleotide sequence. In particular, downstream nucleotidesequences generally relate to sequences that follow the starting pointof transcription. For example, the translation initiation codon of agene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′to a reference nucleotide sequence. In particular, upstream nucleotidesequences generally relate to sequences that are located on the 5′ sideof a coding sequence or starting point of transcription. For example,most promoters are located upstream of the start site of transcription.

The terms “restriction endonuclease” and “restriction enzyme” are usedinterchangeably and refer to an enzyme that binds and cuts within aspecific nucleotide sequence within double stranded DNA.

“Homologous recombination” refers to the insertion of a foreign DNAsequence into another DNA molecule, e.g., insertion of a vector in achromosome. Preferably, the vector targets a specific chromosomal sitefor homologous recombination. For specific homologous recombination, thevector will contain sufficiently long regions of homology to sequencesof the chromosome to allow complementary binding and incorporation ofthe vector into the chromosome. Longer regions of homology, and greaterdegrees of sequence similarity, may increase the efficiency ofhomologous recombination.

Several methods known in the art may be used to propagate apolynucleotide according to the invention. Once a suitable host systemand growth conditions are established, recombinant expression vectorscan be propagated and prepared in quantity. As described herein, theexpression vectors which can be used include, but are not limited to,the following vectors or their derivatives: human or animal viruses suchas vaccinia virus or adenovirus; insect viruses such as baculovirus;yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid andcosmid DNA vectors, to name but a few.

A “vector” refers to any vehicle for the cloning of and/or transfer of anucleic acid into a host cell. A vector may be a replicon to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” refers to any genetic element(e.g., plasmid, phage, cosmid, chromosome, virus) that functions as anautonomous unit of DNA replication in vivo, i.e., capable of replicationunder its own control. The term “vector” includes both viral andnonviral vehicles for introducing the nucleic acid into a cell in vitro,ex vivo or in vivo. A large number of vectors known in the art may beused to manipulate nucleic acids, incorporate response elements andpromoters into genes, etc. Possible vectors include, for example,plasmids or modified viruses including, for example bacteriophages suchas lambda derivatives, or plasmids such as pBR322 or pUC plasmidderivatives, or the Bluescript vector. Another example of vectors thatare useful in the invention is the UltraVector™ Production System(Intrexon Corp., Blacksburg, Va.) as described in WO 2007/038276 and US2004/185556. For example, the insertion of the DNA fragmentscorresponding to response elements and promoters into a suitable vectorcan be accomplished by ligating the appropriate DNA fragments into achosen vector that has complementary cohesive termini. Alternatively,the ends of the DNA molecules may be enzymatically modified or any sitemay be produced by ligating nucleotide sequences (linkers) into the DNAtermini. Such vectors may be engineered to contain selectable markergenes that provide for the selection of cells that have incorporated themarker into the cellular genome. Such markers allow identificationand/or selection of host cells that incorporate and express the proteinsencoded by the marker.

Viral vectors, and particularly retroviral vectors, have been used in awide variety of gene delivery applications in cells, as well as livinganimal subjects. Viral vectors that can be used include, but are notlimited to, retrovirus, adeno-associated virus, pox, baculovirus,vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, andcaulimovirus vectors. Non-viral vectors include plasmids, liposomes,electrically charged lipids (cytofectins), DNA-protein complexes, andbiopolymers. In addition to a nucleic acid, a vector may also compriseone or more regulatory regions, and/or selectable markers useful inselecting, measuring, and monitoring nucleic acid transfer results(transfer to which tissues, duration of expression, etc.).

The term “plasmid” refers to an extra-chromosomal element often carryinga gene that is not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA molecules. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear, circular, orsupercoiled, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3′ untranslated sequence into a cell.

A “cloning vector” refers to a “replicon,” which is a unit length of anucleic acid, preferably DNA, that replicates sequentially and whichcomprises an origin of replication, such as a plasmid, phage or cosmid,to which another nucleic acid segment may be attached so as to bringabout the replication of the attached segment. Cloning vectors may becapable of replication in one cell type and expression in another(“shuttle vector”). Cloning vectors may comprise one or more sequencesthat can be used for selection of cells comprising the vector and/or oneor more multiple cloning sites for insertion of sequences of interest.

The term “expression vector” refers to a vector, plasmid or vehicledesigned to enable the expression of an inserted nucleic acid sequence.The cloned gene, i.e., the inserted nucleic acid sequence, is usuallyplaced under the control of control elements such as a promoter, aminimal promoter, an enhancer, or the like. Initiation control regionsor promoters, which are useful to drive expression of a nucleic acid inthe desired host cell are numerous and familiar to those skilled in theart. Virtually any promoter capable of driving expression of these genescan be used in an expression vector, including but not limited to, viralpromoters, bacterial promoters, animal promoters, mammalian promoters,synthetic promoters, constitutive promoters, tissue specific promoters,pathogenesis or disease related promoters, developmental specificpromoters, inducible promoters, light regulated promoters; CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TP1, alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);3-lactamase, lac, ara, tet, trp, lP_(L), lP_(R), T7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, cauliflower mosaicvirus 35S, CMV 35S minimal, cassava vein mosaic virus (CsVMV),chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase,shoot-specific, root specific, chitinase, stress inducible, rice tungrobacilliform virus, plant super-promoter, potato leucine aminopeptidase,nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin,zein protein, and anthocyanin promoters (useful for expression in plantcells); animal and mammalian promoters known in the art including, butare not limited to, the SV40 early (SV40e) promoter region, the promotercontained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus(RSV), the promoters of the E1A or major late promoter (MLP) genes ofadenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpessimplex virus (HSV) thymidine kinase (TK) promoter, a baculovirus IE1promoter, an elongation factor 1 alpha (EF1) promoter, aphosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, analbumin promoter, the regulatory sequences of the mousemetallothionein-L promoter and transcriptional control regions, theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (of theMDR, CFTR or factor VIII type, and the like), pathogenesis or diseaserelated-promoters, and promoters that exhibit tissue specificity andhave been utilized in transgenic animals, such as the elastase I genecontrol region which is active in pancreatic acinar cells; insulin genecontrol region active in pancreatic beta cells, immunoglobulin genecontrol region active in lymphoid cells, mouse mammary tumor viruscontrol region active in testicular, breast, lymphoid and mast cells;albumin gene, Apo AI and Apo AII control regions active in liver,alpha-fetoprotein gene control region active in liver, alpha1-antitrypsin gene control region active in the liver, beta-globin genecontrol region active in myeloid cells, myelin basic protein genecontrol region active in oligodendrocyte cells in the brain, myosinlight chain-2 gene control region active in skeletal muscle, andgonadotropic releasing hormone gene control region active in thehypothalamus, pyruvate kinase promoter, villin promoter, promoter of thefatty acid binding intestinal protein, promoter of the smooth musclecell α-actin, and the like. In addition, these expression sequences maybe modified by addition of enhancer or regulatory sequences and thelike.

Vectors may be introduced into the desired host cells by methods knownin the art, e.g., transfection, electroporation, microinjection,transduction, cell fusion, DEAE dextran, calcium phosphateprecipitation, lipofection (lysosome fusion), use of a gene gun, or aDNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963(1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al.,Canadian Patent Application No. 2,012,311).

A vector of the invention may also be administered to a subject by anyroute of administration, including, but not limited to, intramuscularadministration.

A polynucleotide according to the invention can also be introduced invivo by lipofection. For the past decade, there has been increasing useof liposomes for encapsulation and transfection of nucleic acids invitro. Synthetic cationic lipids designed to limit the difficulties anddangers encountered with liposome-mediated transfection can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Felgner et al., Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey etal., Proc. Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al.,Science 259:1745 (1993)). The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes (Felgner et al., Science337:387 (1989)). Particularly useful lipid compounds and compositionsfor transfer of nucleic acids are described in WO95/18863, WO96/17823and U.S. Pat. No. 5,459,127. The use of lipofection to introduceexogenous genes into the specific organs in vivo has certain practicaladvantages. Molecular targeting of liposomes to specific cellsrepresents one area of benefit. It is clear that directing transfectionto particular cell types would be particularly preferred in a tissuewith cellular heterogeneity, such as pancreas, liver, kidney, and thebrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting (Mackey et al. 1988, supra). Targeted peptides,e.g., hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as a naked DNA plasmid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther. 3:147 (1992); and Wu et al., J. Biol. Chem.262:4429 (1987)).

The term “transfection” refers to the uptake of exogenous orheterologous RNA or DNA by a cell. A cell has been “transfected” byexogenous or heterologous RNA or DNA when such RNA or DNA has beenintroduced inside the cell. A cell has been “transformed” by exogenousor heterologous RNA or DNA when the transfected RNA or DNA effects aphenotypic change. The transforming RNA or DNA can be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

In addition, the recombinant vector comprising a polynucleotideaccording to the invention may include one or more origins forreplication in the cellular hosts in which their amplification or theirexpression is sought, markers or selectable markers.

The term “selectable marker” refers to an identifying factor, usually anantibiotic or chemical resistance gene, that is able to be selected forbased upon the marker gene's effect, i.e., resistance to an antibiotic,resistance to a herbicide, colorimetric markers, enzymes, fluorescentmarkers, and the like, wherein the effect is used to track theinheritance of a nucleic acid of interest and/or to identify a cell ororganism that has inherited the nucleic acid of interest. Examples ofselectable marker genes known and used in the art include: genesproviding resistance to ampicillin, streptomycin, gentamycin, kanamycin,hygromycin, bialaphos herbicide, sulfonamide, and the like; and genesthat are used as phenotypic markers, i.e., anthocyanin regulatory genes,isopentanyl transferase gene, and the like.

The term “reporter gene” refers to a nucleic acid encoding anidentifying factor that is able to be identified based upon the reportergene's effect, wherein the effect is used to track the inheritance of anucleic acid of interest, to identify a cell or organism that hasinherited the nucleic acid of interest, and/or to measure geneexpression induction or transcription. Examples of reporter genes knownand used in the art include: luciferase (Luc), green fluorescent protein(GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ),β-glucuronidase (Gus), and the like. Selectable marker genes may also beconsidered reporter genes.

“Promoter” and “promoter sequence” are used interchangeably and refer toa DNA sequence capable of controlling the expression of a codingsequence or functional RNA. In general, a coding sequence is located 3′to a promoter sequence. Promoters may be derived in their entirety froma native gene, or be composed of different elements derived fromdifferent promoters found in nature, or even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological conditions. Promoters thatcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters.” Promoters that cause agene to be expressed in a specific cell type are commonly referred to as“cell-specific promoters” or “tissue-specific promoters.” Promoters thatcause a gene to be expressed at a specific stage of development or celldifferentiation are commonly referred to as “developmentally-specificpromoters” or “cell differentiation-specific promoters.” Promoters thatare induced and cause a gene to be expressed following exposure ortreatment of the cell with an agent, biological molecule, chemical,ligand, light, or the like that induces the promoter are commonlyreferred to as “inducible promoters” or “regulatable promoters.” It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

In any of the vectors of the present invention, the vector optionallycomprises a promoter disclosed herein. In one embodiment, the promoteris a promoter listed in Table 1 herein.

In any of the vectors of the present invention, the vector optionallycomprises a tissue-specific promoter. In one embodiment, thetissue-specific promoter is a tissue specific promoter disclosed herein.In another embodiment, the tissue-specific promoter is a tissue specificpromoter listed in Table 2 herein.

The promoter sequence is typically bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence is found a transcription initiation site (conveniently definedfor example, by mapping with nuclease S1), as well as protein bindingdomains (consensus sequences) responsible for the binding of RNApolymerase.

“Therapeutic switch promoter” (“TSP”) refers to a promoter that controlsexpression of a gene switch component. See, for example, US2009/0098055, which is hereby incorporated by reference in its entirety.Gene switches and their various components are described in detailelsewhere herein. In certain embodiments a TSP is constitutive, i.e.,continuously active. A constitutive TSP may be eitherconstitutive-ubiquitous (i.e., generally functions, without the need foradditional factors or regulators, in any tissue or cell) orconstitutive-tissue or cell specific (i.e., generally functions, withoutthe need for additional factors or regulators, in a specific tissue typeor cell type). In certain embodiments a TSP of the invention isactivated under conditions associated with a disease, disorder, orcondition. In certain embodiments of the invention where two or moreTSPs are involved the promoters may be a combination of constitutive andactivatable promoters. As used herein, a “promoter activated underconditions associated with a disease, disorder, or condition” includes,without limitation, disease-specific promoters, promoters responsive toparticular physiological, developmental, differentiation, orpathological conditions, promoters responsive to specific biologicalmolecules, and promoters specific for a particular tissue or cell typeassociated with the disease, disorder, or condition, e.g. tumor tissueor malignant cells. TSPs can comprise the sequence of naturallyoccurring promoters, modified sequences derived from naturally occurringpromoters, or synthetic sequences (e.g., insertion of a response elementinto a minimal promoter sequence to alter the responsiveness of thepromoter).

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced (if the coding sequence contains introns) and translated intothe protein encoded by the coding sequence.

“Transcriptional and translational control sequences” refer to DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

The term “response element” refers to one or more cis-acting DNAelements which confer responsiveness on a promoter mediated throughinteraction with the DNA-binding domains of a transcription factor. ThisDNA element may be either palindromic (perfect or imperfect) in itssequence or composed of sequence motifs or half sites separated by avariable number of nucleotides. The half sites can be similar oridentical and arranged as either direct or inverted repeats or as asingle half site or multimers of adjacent half sites in tandem. Theresponse element may comprise a minimal promoter isolated from differentorganisms depending upon the nature of the cell or organism into whichthe response element is incorporated. The DNA binding domain of thetranscription factor binds, in the presence or absence of a ligand, tothe DNA sequence of a response element to initiate or suppresstranscription of downstream gene(s) under the regulation of thisresponse element. Examples of DNA sequences for response elements of thenatural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 1)(see Cherbas et. al., Genes Dev. 5:120 (1991)); AGGTCAN(n)AGGTCA (SEQ IDNO: 2), where N_((n)) can be one or more spacer nucleotides (see D'Avinoet al., Mol. Cell. Endocrinol. 113:1 (1995)); and GGGTTGAATGAATTT (SEQID NO: 3) (see Antoniewski et al., Mol. Cell Biol. 14:4465 (1994)).

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid or polynucleotide. Expression may also refer to translationof mRNA into a protein or polypeptide.

The terms “cassette,” “expression cassette” and “gene expressioncassette” refer to a segment of DNA that can be inserted into a nucleicacid or polynucleotide at specific restriction sites or by homologousrecombination. The segment of DNA comprises a polynucleotide thatencodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. “Transformationcassette” refers to a specific vector comprising a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.Cassettes, expression cassettes, gene expression cassettes andtransformation cassettes of the invention may also comprise elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

For purposes of this invention, the term “gene switch” refers to thecombination of a response element associated with a promoter, and aligand-dependent transcription factor-based system which, in thepresence of one or more ligands, modulates the expression of a gene intowhich the response element and promoter are incorporated. The term “apolynucleotide encoding a gene switch” refers to the combination of aresponse element associated with a promoter, and a polynucleotideencoding a ligand-dependent transcription factor-based system which, inthe presence of one or more ligands, modulates the expression of a geneinto which the response element and promoter are incorporated.

The therapeutic switch promoters of the invention may be any promoterthat is useful for treating, ameliorating, or preventing a specificdisease, disorder, or condition. Examples include, without limitation,promoters of genes that exhibit increased expression only during aspecific disease, disorder, or condition and promoters of genes thatexhibit increased expression under specific cell conditions (e.g.,proliferation, apoptosis, change in pH, oxidation state, oxygen level).In some embodiments where the gene switch comprises more than onetranscription factor sequence, the specificity of the therapeuticmethods can be increased by combining a disease- or condition-specificpromoter with a tissue- or cell type-specific promoter to limit thetissues in which the therapeutic product is expressed. Thus, tissue- orcell type-specific promoters are encompassed within the definition oftherapeutic switch promoter.

As an example of disease-specific promoters, useful promoters fortreating cancer include the promoters of oncogenes, including promotersfor treating anemia. Examples of classes of oncogenes include, but arenot limited to, growth factors, growth factor receptors, proteinkinases, programmed cell death regulators and transcription factors.Specific examples of oncogenes include, but are not limited to, sis, erbB, erb B-2, ras, abl, myc and bcl-2 and TERT. Examples of othercancer-related genes include tumor associated antigen genes and othergenes that are overexpressed in neoplastic cells (e.g., MAGE-1,carcinoembryonic antigen, tyrosinase, prostate specific antigen,prostate specific membrane antigen, p53, MUC-1, MUC-2, MUC-4, HER-2/neu,T/Tn, MART-1, gp100, GM2, Tn, sTn, and Thompson-Friedenreich antigen(TF)).

Examples of promoter sequences and other regulatory elements (e.g.,enhancers) that are known in the art and are useful as therapeuticswitch promoters in the present invention are disclosed in thereferences listed in Tables 1 and 2, along with the disease/disorder(Table 1) or tissue specificity (Table 2) associated with each promoter.The promoter sequences disclosed in the U.S. patents and published U.S.applications cited in the Tables and the sequences disclosed therein areherein incorporated by reference in their entirety.

The polynucleotide encoding any of the proteins listed in Table 1 mayalso be expressed using a vector of the present invention with apromoter that is not a therapeutic promoter.

TABLE 1 Patent/Published Promoter Sequence Disease/Disorder ApplicationNo. Her-2/neu (ERBB2/c-erbB-2) cancer 5,518,885 Osteocalcin calcifiedtumors 5,772,993 stromelysin-1 cancer 5,824,794 prostate specificantigen prostate cancer 5,919,652 human sodium-iodide symporter thyroidcarcinoma 6,015,376 H19, IF-1, IGF-2 cancer 6,306,833 thymosin β15breast, pancreatic, prostate 6,489,463 cancer T cell factor cancer6,608,037 cartilage-derived retinoic acid- chondrosarcoma, 6,610,509sensitive protein mammary tumor Insulin pancreatic cancer 6,716,824PEG-3 cancer 6,737,523 telomerase reverse transcriptase cancer 6,777,203melanoma differentiation associated cancer 6,841,362 gene-7 Prostasincancer 6,864,093 telomerase catalytic subunit; cancer 6,936,595 cyclin-Amidkine; c-erbB-2 cancer 7,030,099 prostate-specific membrane antigenprostate cancer 7,037,647 p51 cancer 7,038,028 telomerase RNA cancer7,084,267 prostatic acid phosphatase prostate cancer 7,094,533PCA3_(dd3) prostate cancer 7,138,235 DF3/MUC1 cancer 7,247,297 hex IIcancer 2001/0011128 cyclooxygenase-2 cancer 2002/0107219 super PSAprostate cancer 2003/0078224 skp2 cancer 2003/0109481 PRL-3 metastaticcolon cancer 2004/0126785 CA125/M17S2 ovarian cancer 2004/0126824 IAI.3Bovarian cancer 2005/0031591 CRG-L2 liver cancer 2005/0124068 TRPM4prostate cancer 2006/0188990 RTVP glioma 2006/0216731 TARP prostatecancer, breast 2007/0032439 cancer telomere reverse transcriptase cancer2007/0059287 A4 amyloid protein Alzheimer's disease 5,151,508 amyloidβ-protein precursor Alzheimer's disease 5,643,726 precursor of theAlzheimer's Disease Alzheimer's disease 5,853,985 A4 amyloid proteinneuropeptide FF CNS disorders 6,320,038 endoplasmic reticulum stressstress 7,049,132 elements urocortin II psychopathologies 7,087,385tyrosine hydroxylase neurological disorders 7,195,910 complement factor3; serum amyloid inflammation 5,851,822 A3 tissue inhibitor ofmetalloproteinase- rheumatism, cancer, 5,854,019 3 (TIMP-3) autoimmunedisease, inflammation p75 tumor necrosis factor receptor autoimmunedisease 5,959,094 tumor necrosis factor-α inflammation 6,537,784peroxisome proliferator activated inflammation 6,870,044 receptor/IIA-1nonpancreatic secreted phospholipase A2 SOCS-3 growth disorders,2002/0174448 autoimmune disease, inflammation SR-BI lipid disorders5,965,790 Ob obesity 5,698,389 site-1 protease obesity, diabetes7,045,294 TIGR glaucoma 7,138,511 VL30 anoxia 5,681,706 excitatory aminoacid transporter-2 nervous system ischemia 2004/0171108 MDTS9 renalfailure 2006/0014931 LIM, pyrroline 5-carboxylate prostate disorders2006/0134688 reductase, SIM2 Bax apoptosis 5,744,310 Fas apoptosis5,888,764 bbc3 apoptosis 7,202,024 PINK-1 PI-3 kinase/Akt pathway2006/0228776 disorders

TABLE 2 Patent/Published Promoter Sequence Tissue SpecificityApplication No. troponin T skeletal muscle 5,266,488 myoD muscle5,352,595 Actin muscle 5,374,544 smooth muscle 22α arterial smoothmuscle 5,837,534 Utrophin muscle 5,972,609 Myostatin muscle 6,284,882smooth muscle myosin heavy chain smooth muscle 6,780,610 cardiac ankyrinrepeat protein cardiac muscle 7,193,075 MLP muscle 2002/0042057Smoothelin smooth muscle 2003/0157494 MYBPC3 cardiomyocytes 2004/0175699Tα1 α-tabulin neurons 5,661,032 intercellular adhesion molecule-4neurons 5,753,502 (ICAM-4) γ-aminobutyric acid type A receptorhippocampus 6,066,726 β1 subunit neuronal nicotinic acetylcholineneurons 6,177,242 receptor β2-subunit presenilin-1 neurons 6,255,473calcium-calmodulin-dependent forebrain 6,509,190 kinase IIα CRF_(2α)receptor brain 7,071,323 nerve growth factor neurons 2003/159159  GLP-2receptor gut, brain 2002/0045173 type I transglutaminase keratinocytes5,643,746 K14 keratinocytes 6,596,515 stearoyl-CoA desaturase skin2002/0151018 Megsin renal cells 6,790,617 Prolactin pituitary 5,082,779GDF-9 ovary, testes, 7,227,013 hypothalamus, pituitary, placenta PSP94prostate 2003/0110522 NRL; NGAL mammary gland 5,773,290 long whey acidicprotein mammary gland 5,831,141 mammary associated amyloid A mammaryductal epithelial 2005/0107315 cells endothelin-1 endothelial cells5,288,846 Serglycin hematopoietic cells 5,340,739 platelet-endothelialcell adhesion platelets, leukocytes, 5,668,012 molecule-1 (PECAM-1)endothelial cells Tie receptor tyrosine kinase endothelial cells, bone5,877,020 marrow KDR/flk-1 endothelial cells 5,888,765 Endoglinendothelial cells 6,103,527 CCR5 myeloid and lymphoid 6,383,746 cellsCD11d myeloid cells 6,881,834 platelet glycoprotein IIb hematopoieticcells 6,884,616 preproendothelin-1 endothelial cells 7,067,649interleukin-18 binding protein mononuclear cells 2006/0239984 CD34hematopoietic stem cells 5,556,954 Tec tyrosine kinase hematopoieticstem cells, 6,225,459 liver

Other genes that exhibit changes in expression levels during specificdiseases or disorders and therefore may provide promoters that areuseful in the present invention include, without limitation, the genes(along with the associated disease/disorder) listed in Table 3.

TABLE 3 Patent/Published Gene Disease/Disorder Application No. MLH1,MSH2, MSH6, PMS1, APC Colorectal cancer 7,148,016 LEF-1 Colon cancer2002/6169300 F₂ receptor Colon cancer 2002/0187502 TGF-β type IIreceptor Colon cancer 2004/0038284 EYA4 Colon cancer 2005/0003463 PCA3Prostate cancer 7,138,235 K2 Prostate cancer 6,303,361 PROST 03 Prostatecancer metastases 2002/0009455 PCAM-1 Prostate cancer 2002/0042062PCADM-1 Prostate cancer 2003/0100033 PCA3_(dd3) Prostate cancer2003/0165850 PCAV Prostate cancer 2006/0275747 PAcP Androgen-insensitive2006/0294615 prostate cancer SEQ ID NO: 1 of the patent Liver cancer5,866,329 5,866,329, incorporated by reference herein SEQ ID NOS: 1, 3of the U.S. patent Hepatocellular cancer 2002/0115094 applicationpublication 2002/0115094, incorporated by reference herein SEQ ID NO: 1of the patent U.S. Hepatocellular carcinoma 2005/0037372 applicationpublication 2005/0037372, incorporated by reference herein ATB₀Hepatocellular carcinoma 2006/0280725 SEQ ID NOS: 1, 3 of the U.S.patent Liver cancer 2007/0042420 application publication 2007/0042420CSA-1 Chondrosarcoma 2001/0016649 SEQ ID NOS: 1-15 of the U.S. patentPancreatic cancer 2001/0016651 application publication 2001/0016651,incorporated by reference herein SEQ ID NOS: 1-15 of the U.S. patentPancreatic cancer 2003/0212264 application publication 2003/0212264,incorporated by reference herein SYG972 Breast cancer 2002/0055107Urb-ctf Breast cancer 2003/0143546 BCU399 Breast cancer 2003/0180728TBX2 Breast cancer 2004/0029185 Cyr61 Breast cancer 2004/0086504 DIAPH3Breast cancer 2005/0054826 SEQ ID NOS: 1-24 of the U.S. patent Breastcancer 2007/0134669 application publication 2007/0134669, incorporatedby reference herein Human aspartyl (asparaginyl) beta- CNS cancer2002/0102263 hydroxylase BEHAB CNS cancer 2003/0068661 IL-8 Kaposi'sSarcoma 2003/0096781 SEQ ID NOS: 1-278 of the U.S. Hematological cancers2002/0198362 patent application publication 2002/0198362, incorporatedby reference herein BLSA B-cell cancer 2003/0147887 BP1 Leukemia2003/0171273 DAP-kinase, HOXA9 Non-small cell lung cancer 2003/0224509ARP Clear cell renal carcinoma, 2004/0010119 inflammatory disorders NbkRenal cancer 2005/0053931 CD43 Ovarian cancer 2006/0216231 SEQ ID NOS:1-84 of the U.S. patent Ovarian cancer 2007/0054268 applicationpublication 2007/0054268, incorporated by reference herein β7-hcG,β6-hCG, β6e-hCG, Uterine tumors 2006/0292567 β5-hCG, β8-hcG, β3-hCGMTA1s Hormone insensitive 2006/0204957 cancer Old-35, Old-64 Tumorproliferation 2003/0099660 LAGE-1 Cancer 6,794,131 CIF150/hTAF_(II)150Cancer 6,174,679 P65 oncofetal protein Cancer 5,773,215 TelomeraseCancer 2002/0025518 CYP1B1 Cancer 2002/0052013 14-3-3σ Cancer2002/0102245 NES1 Cancer 2002/0106367 CAR-1 Cancer 2002/0119541 HMGI,MAG Cancer 2002/0120120 ELL2 Cancer 2002/0132329 Ephrin B2 Cancer2002/0136726 WAF1 Cancer 2002/0142442 CIF130 Cancer 2002/0143154 C35Cancer 2002/0155447 BMP2 Cancer 2002/0159986 BUB3 Cancer 2002/0160403Polymerase kappa Cancer 2003/0017573 EAG1, EAG2 Cancer 2003/0040476 SEQID NOS: 18, 20, 22 of the U.S. Cancer 2003/0044813 patent applicationpublication 2003/0044813, incorporated by reference herein HMGI Cancer2003/0051260 HLTF Cancer 2003/0082526 Barx2 Cancer 2003/0087243 SEQ IDNOS: 18, 20, 22, 32, 34, Cancer 2003/0108920 36 of the U.S. patentapplication publication 2003/0108920, incorporated by reference hereinCables Cancer 2003/0109443 Pp 32r1 Cancer 2003/0129631 BMP4 Cancer2003/0134790 TS10q23.3 Cancer 2003/0139324 Nuclear spindle-associatingprotein Cancer 2003/0157072 PFTAIRE Cancer 2003/0166217 SEMA3B Cancer2003/0166557 MOGp Cancer, multiple sclerosis, 2003/0166898 inflammatorydisease Fortilin Cancer 2003/0172388 SEQ ID NO: 1 of the U.S. patentCancer 2003/0215833 application publication 2003/0215833, incorporatedby reference herein IGFBP-3 Cancer 2004/0005294 Polyhomeotic 2 Cancer2004/0006210 PNQALRE Cancer 2004/0077009 SEQ ID NOS: 1, 3 of the U.S.patent Cancer 2004/0086916 application publication 2004/0086916,incorporated by reference herein SCN5A Cancer 2004/0146877 miR15, miR16Cancer 2004/0152112 Headpin Cancer 2004/0180371 PAOh1/SMO Cancer2004/0229241 Hippo, Mst2 Cancer 2005/0053592 PSMA-like Cancer,neurological 2005/0064504 disorders JAB1 Cancer 2005/0069918 NF-ATCancer 2005/0079496 P28ING5 Cancer 2005/0097626 MTG16 Cancer2005/0107313 ErbB-2 Cancer 2005/0123538 HDAC9 Cancer 2005/0130146 GPBPCancer 2005/0130227 MG20 Cancer 2005/0153352 KLF6 Cancer 2005/0181374ARTS1 Cancer 2005/0266443 Dock 3 Cancer 2006/0041111 Annexin 8 Cancer2006/0052320 MH15 Cancer 2006/0068411 DELTA-N p73 Cancer 2006/0088825RapR6 Cancer 2006/099676 StarD10 Cancer 2006/0148032 Ciz1 Cancer2006/0155113 HLJ1 Cancer 2006/0194235 RapR7 Cancer 2006/0240021 A34Cancer 2006/0292154 Sef Cancer 2006/0293240 Killin Cancer 2007/0072218SGA-1M Cancer 2007/0128593 TGFβ Type II receptor Cancer 2002/0064786GCA-associated genes Giant cell arteritis 6,743,903 PRV-1 Polycythemiavera 6,686,153 SEQ ID NOS: 2, 4 of the U.S. patent Ischemia 5,948,6375,948,637, incorporated by reference herein Vezf1 Vascular disorders2002/0023277 MLP Dilatative cardiomyopathy 2002/0042057 VEGIPathological angiogenesis 2002/0111325 PRO256 Cardiovascular disorders2002/0123091 AOP2 Atherosclerosis 2002/0142417 Remodelin Arterialrestenosis, fibrosis 2002/0161211 Phosphodiesterase 4D Stroke2003/0054531 Prostaglandin receptor subtype EP3 Peripheral arterial2003/0157599 occlusive disease CARP Heart disorders 2004/0014706 HOPCongenital heart disease 2004/0029158 SEQ ID NOS: 1-4 of the U.S. patentApoplexy 2004/0087784 application publication 2004/0087784, incorporatedby reference herein PLTP Atherosclerosis, vascular 2006/0252787 disease,hypercholesterolemia, Tangier's disease, familial HDL deficiency diseaseSEQ ID NOS: 1, 3-8, 15, 16 of the Thrombosis 2007/0160996 U.S. patentapplication publication 2007/0160996, incorporated by reference hereinUCP-2 Stroke 2002/0172958 FLJ11011 Fanconi's Anemia 2006/0070134Codanin-1 Anemia 2006/0154331 SEQ ID NOS: 1, 6, 8 of the U.S.Insulin-dependent diabetes 5,763,591 Pat. No. 5,763,591, incorporated bymellitus reference herein Resistin Type II diabetes 2002/0161210Archipelin Diabetes 2003/0202976 SEQ ID NOS: 2, 7, 16, 27 of the U.S.Diabetes, hyperlipidemia 2004/0053397 patent application publication2004/0053397, incorporated by reference herein Neuronatin Metabolicdisorders 2004/0259777 Ncb5or Diabetes 2005/0031605 7B2 Endocrinedisorders 2005/0086709 PTHrP, PEX Metabolic bone diseases 2005/0113303KChIP1 Type II diabetes 2005/0196784 SLIT-3 Type II diabetes2006/0141462 CX3CR1 Type II diabetes 2006/0160076 SMAP-2 Diabetes2006/0210974 SEQ ID NOS: 2, 8, 12, 16, 22, 26, Type II diabetes2006/0228706 28, 32 of the U.S. patent application publication2006/0228706, incorporated by reference herein IC-RFX Diabetes2006/0264611 E2IG4 Diabetes, insulin 2007/0036787 resistance, obesitySEQ ID NOS: 2, 8, 10, 14, 18, 24, Diabetes 2007/0122802 26, 30, 34, 38,44, 50, 54, 60, 62, 68, 74, 80, 86, 92, 98, 104, 110 of the U.S. patentapplication publication 2007/0122802, incorporated by reference hereinUCP2 Body weight disorders 2002/0127600 Ob receptor Body weightdisorders 2002/0182676 Ob Bodyweight disorders 2004/0214214 Dp1Neurodegenerative 2001/0021771 disorders NRG-1 Schizophrenia2002/0045577 Synapsin III Schizophrenia 2002/0064811 NRG1AG1Schizophrenia 2002/0094954 AL-2 Neuronal disorders 2002/0142444 Prolinedehydrogenase Bipolar disorder, major 2002/0193581 depressive disorder,schizophrenia, obsessive compulsive disorder MNR2 Chronicneurodegenerative 2002/0197678 disease ATM Ataxia-telangiectasia2004/0029198 Ho-1 Dementing diseases 2004/0033563 CON202 Schizophrenia2004/0091928 Ataxin-1 Neurodegenerative 2004/0177388 disorders NR3BMotor neuron disorders 2005/0153287 NIPA-1 Hereditary spastic2005/0164228 paraplegia DEPP, adrenomedullin, csdA Schizophrenia2005/0227233 Inf-20 Neurodegenerative 2006/0079675 diseases EOPA Braindevelopment and 2007/0031830 degeneration disorders SERT Autism2007/0037194 FRP-1 Glaucoma 2002/0049177 Serum amyloid A Glaucoma2005/0153927 BMP2 Osteoporosis 2002/0072066 BMPR1A Juvenile polyposis2003/0072758 ACLP Gastroschisis 2003/0084464 Resistin-like molecule βFamilial adenomatous 2003/0138826 polyposis, diabetes, insulinresistance, colon cancer, inflammatory bowel disorder Dlg5 Inflammatorybowel 2006/0100132 disease SEQ ID NOS: 1-82 of the U.S. patentOsteoarthritis 2002/0119452 application publication 2002/0119452,incorporated by reference herein TRANCE Immune system disorders2003/0185820 Matrilin-3 Osteoarthritis 2003/0203380 SynoviolinRheumatoid arthritis 2004/0152871 SEQ ID NOS: 9, 35 of the U.S.Osteoarthritis 2007/0028314 patent application publication 2007/0028314,incorporated by reference herein HIV LTR HIV infection 5,627,023 SHIVAHIV infection 2004/0197770 EBI 1, EBI 2, EBI 3 Epstein Barr virusinfection 2002/0040133 NM23 family Skin/intestinal disorders2002/0034741 SEQ ID NO: 1 of the U.S. patent Psoriasis 2002/0169127application publication 2002/0169127, incorporated by reference hereinEps8 Skin disorders, wound 2003/0180302 healing Beta-10 Thyroid glandpathology 2002/0015981 SEQ ID NO: 2 of the U.S. patent Thyroidconditions 2003/0207403 application publication 2003/0207403,incorporated by reference herein SEQ ID NO: 3 of the U.S. patent Thyroiddisorders 2007/0020275 application publication 2007/0020275,incorporated by reference herein Hair follicle growth factor Alopecia2003/0036174 Corneodesmosin Alopecia 2003/0211065 GCR9 Asthma, lymphoma,2003/0166150 leukemia SEQ ID NO: 1-71 of the U.S. patent Asthma2004/0002084 application publication 2004/0002084, incorporated byreference herein Bg Chediak-Higashi syndrome 2002/0115144 SEQ ID NOS:1-16 of the U.S. patent Endometriosis 2002/0127555 applicationpublication 2002/0127555, incorporated by reference herein FGF23Hypophosphatemic 2005/0156014 disorders BBSR Bardet-Biedl syndrome2003/0152963 MIC-1 Fetal abnormalities, cancer, 2004/0053325inflammatory disorders, miscarriage, premature birth MIA-2 Liver damage2004/0076965 IL-17B Cartilage degenerative 2004/0171109 disordersFormylglycine generating enzyme Multiple sulfatase 2004/0229250deficiency LPLA2 Pulmonary alveolar 2006/0008455 proteinosis CXCL1ORespiratory illnesses 2006/0040329 SEQ ID NOS: 1, 2 of the U.S. patentNephropathy 2006/0140945 application publication 2006/0140945,incorporated by reference herein HFE2A Iron metabolism disease2007/0166711

Once a gene with an expression pattern that is modulated during adisease, disorder, or condition is identified, the promoter of the genemay be used in the gene switch of the invention. The sequence of manygenes, including the promoter region, is known in the art and availablein public databases, e.g., GenBank. Thus, once an appropriate gene isidentified, the promoter sequence can be readily identified andobtained. Another aspect of the present invention is directed towardsidentifying suitable genes whose promoter can be isolated and placedinto a gene switch. The identity of the gene, therefore, may not becritical to specific embodiments of the present invention, provided thepromoter can be isolated and used in subsequent settings orenvironments. The current invention thus includes the use of promotersfrom genes that are yet to be identified. Once suitable genes areidentified, it is a matter of routine skill or experimentation todetermine the genetic sequences needed for promoter function. Indeed,several commercial protocols exist to aid in the determination of thepromoter region of genes of interest. By way of example, Ding et al.recently elucidated the promoter sequence of the novel Sprouty4 gene(Am. J. Physiol. Lung Cell. Mol. Physiol. 287: L52 (2004), which isincorporated by reference) by progressively deleting the 5′-flankingsequence of the human Sprouty4 gene. Briefly, once the transcriptioninitiation site was determined, PCR fragments were generated usingcommon PCR primers to clone segments of the 5′-flanking segment in aunidirectional manner. The generated segments were cloned into aluciferase reporter vector and luciferase activity was measured todetermine the promoter region of the human Sprouty4 gene.

Another example of a protocol for acquiring and validating genepromoters includes the following steps: (1) acquire diseased andnon-diseased cell/tissue samples of similar/same tissue type; (2)isolate total RNA or mRNA from the samples; (3) perform differentialmicroarray analysis of diseased and non-diseased RNA; (4) identifycandidate disease-specific transcripts; (5) identify genomic sequencesassociated with the disease-specific transcripts; (6) acquire orsynthesize DNA sequence upstream and downstream of the predictedtranscription start site of the disease-specific transcript; (7) designand produce promoter reporter vectors using different lengths of DNAfrom step 6; and (8) test promoter reporter vectors in diseased andnon-diseased cells/tissues, as well as in unrelated cells/tissues.

The source of the promoter that is inserted into the gene switch can benatural or synthetic, and the source of the promoter should not limitthe scope of the invention described herein. In other words, thepromoter may be directly cloned from cells, or the promoter may havebeen previously cloned from a different source, or the promoter may havebeen synthesized.

In another embodiment, a polynucleotide encoding any of gene productsreferred to in Tables 1-3 may be used in the vector and methods of thepresent invention, for therapeutic uses and for diagnostic purposes.

Gene Switch Systems

The gene switch may be any gene switch that regulates gene expression byaddition or removal of a specific ligand. In one embodiment, the geneswitch is one in which the level of gene expression is dependent on thelevel of ligand that is present. Examples of ligand-dependenttranscription factor complexes that may be used in the gene switches ofthe invention include, without limitation, members of the nuclearreceptor superfamily activated by their respective ligands (e.g.,glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs andmimetics thereof) and rTTA activated by tetracycline. In one aspect ofthe invention, the gene switch is an EcR-based gene switch. Examples ofsuch systems include, without limitation, the systems described in U.S.Pat. Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos.2006/0014711, 2007/0161086, and International Published Application No.WO 01/70816. Examples of chimeric ecdysone receptor systems aredescribed in U.S. Pat. No. 7,091,038, U.S. Published Patent ApplicationNos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and2006/0100416, and International Published Application Nos. WO 01/70816,WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, andWO 2005/108617, each of which is incorporated by reference in itsentirety. An example of a non-steroidal ecdysone agonist-regulatedsystem is the RheoSwitch® Mammalian Inducible Expression System (NewEngland Biolabs, Ipswich, Mass.). In another aspect of the invention,the gene switch is based on heterodimerization of FK506 binding protein(FKBP) with FKBP rapamycin associated protein (FRAP) and is regulatedthrough rapamycin or its non-immunosuppressive analogs. Examples of suchsystems, include, without limitation, the ARGENT™ TranscriptionalTechnology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systemsdescribed in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757,and 6,649,595.

In one embodiment, the gene switch comprises a single transcriptionfactor sequence encoding a ligand-dependent transcription factor complexunder the control of a therapeutic switch promoter. The transcriptionfactor sequence may encode a ligand-dependent transcription factorcomplex that is a naturally occurring or an artificial ligand-dependenttranscription factor complex. An artificial transcription factor is onein which the natural sequence of the transcription factor has beenaltered, e.g., by mutation of the sequence or by the combining ofdomains from different transcription factors. In one embodiment, thetranscription factor comprises a Group H nuclear receptor ligand bindingdomain. In one embodiment, the Group H nuclear receptor ligand bindingdomain is from an ecdysone receptor, a ubiquitous receptor (UR), anorphan receptor 1 (OR-1), a steroid hormone nuclear receptor 1 (NER-1),a retinoid X receptor interacting protein-15 (RIP-15), a liver Xreceptor 3 (LXR4), a steroid hormone receptor like protein (RLD-1), aliver X receptor (LXR), a liver X receptor α (LXRα), a farnesoid Xreceptor (FXR), a receptor interacting protein 14 (RIP-14), or afarnesol receptor (HRR-1). In another embodiment, the Group H nuclearreceptor LBD is from an ecdysone receptor.

A. Ecdysone-Based Gene Switch

The EcR and the other Group H nuclear receptors are members of thenuclear receptor superfamily wherein all members are generallycharacterized by the presence of an amino-terminal transactivationdomain (AD, also referred to interchangeably as “TA” or “TD”),optionally fused to a heterodimerization partner (HP) to form acoactivation protein (CAP), a DNA binding domain (DBD), and a LBD fusedto the DBD via a hinge region to form a ligand-dependent transcriptionfactor (LTF). As used herein, the term “DNA binding domain” comprises aminimal polypeptide sequence of a DNA binding protein, up to the entirelength of a DNA binding protein, so long as the DNA binding domainfunctions to associate with a particular response element. Members ofthe nuclear receptor superfamily are also characterized by the presenceof four or five domains: A/B, C, D, E, and in some members F (see U.S.Pat. No. 4,981,784 and Evans, Science 240:889 (1988)). The “A/B” domaincorresponds to the transactivation domain, “C” corresponds to the DNAbinding domain, “D” corresponds to the hinge region, and “E” correspondsto the ligand binding domain. Some members of the family may also haveanother transactivation domain on the carboxy-terminal side of the LBDcorresponding to “F”.

The following polypeptide sequence was reported as a polypeptidesequence of Ecdysone receptor (Ecdysteroid receptor)(20-hydroxy-ecdysone receptor) (20E receptor) (EcRH) (Nuclear receptorsubfamily 1 group H member 1) and has the accession number P34021 inGenbank.

Ecdysone receptor (878aa) from Drosophila melanogaster (Fruit fly) (SEQID NO:5)

1 mkrrwsnngg fmrlpeesss evtsssnglv lpsgvnmsps sldshdycdq dlwlcgnesg 61sfggsnghgl sqqqqsvitl amhgcsstlp aqttiiping nangnggstn gqyvpgatnl 121galangmlng gfngmqqqiq nghglinstt pstpttplhl qqnlggaggg giggmgilhh 181angtpnglig vvgggggvgl gvggggvggl gmqhtprsds vnsissgrdd lspssslngy 241sanescdakk skkgpaprvq eelclvcgdr asgyhynalt cegckgffrr svtksavycc 301kfgracemdm ymrrkcqecr lkkclavgmr pecvvpenqc amkrrekkaq kekdkmttsp 361ssqhggngsl asgggqdfvk keildlmtce ppqhatipll pdeilakcqa rnipsltynq 421laviykliwy qdgyeqpsee dlrrimsqpd enesqtdvsf rhiteitilt vqlivefakg 481lpaftkipqe dqitllkacs sevmmlrmar rydhssdsif fannrsytrd sykmagmadn 541iedllhfcrq mfsmkvdnve yalltaivif sdrpglekaq lveaiqsyyi dtlriyilnr 601hcgdsmslvf yakllsilte lrtlgnqnae mcfslklknr klpkfleeiw dvhaippsvq 661shlqitqeen erleraermr asvggaitag idcdsastsa aaaaaqhqpq pqpqpqpssl 721tqndsqhqtq pqlqpqlppq lqgqlqpqlq pqlqtqlqpq iqpqpqllpv sapvpasvta 781pgslsavsts seymggsaai gpitpattss itaavtasst tsavpmgngv gvgvgvggnv 841smyanaqtam almgvalhsh qeqliggvav ksehstta

In one embodiment, the ecdysone receptor ligand binding domain isselected from the group consisting of an invertebrate ecdysone receptorligand binding domain, an Arthropod ecdysone receptor ligand bindingdomain, a Lepidopteran ecdysone receptor ligand binding domain, aDipteran ecdysone receptor ligand binding domain, an Orthopteranecdysone receptor ligand binding domain, a Homopteran ecdysone receptorligand binding domain, a Hemipteran ecdysone receptor ligand bindingdomain, a spruce budworm Choristoneura fumiferana EcR ecdysone receptorligand binding domain, a beetle Tenebrio molitor ecdysone receptorligand binding domain, a Manduca sexta ecdysone receptor ligand bindingdomain, a Heliothies virescens ecdysone receptor ligand binding domain,a midge Chironomus tentans ecdysone receptor ligand binding domain, asilk moth Bombyx mori ecdysone receptor ligand binding domain, asquinting bush brown Bicyclus anynana ecdysone receptor ligand bindingdomain, a buckeye Junonia coenia ecdysone receptor ligand bindingdomain, a fruit fly Drosophila melanogaster ecdysone receptor ligandbinding domain, a mosquito Aedes aegypti ecdysone receptor ligandbinding domain, a blowfly Lucilia capitata ecdysone receptor ligandbinding domain, a blowfly Lucilia cuprina ecdysone receptor ligandbinding domain, a blowfly Calliphora vicinia ecdysone receptor ligandbinding domain, a Mediterranean fruit fly Ceratitis capitata ecdysonereceptor ligand binding domain, a locust Locusta migratoria ecdysonereceptor ligand binding domain, an aphid Myzus persicae ecdysonereceptor ligand binding domain, a fiddler crab Celuca pugilator ecdysonereceptor ligand binding domain, an ixodid tick Amblyomma americanumecdysone receptor ligand binding domain, a whitefly Bamecia argentifoliecdysone receptor ligand binding domain and a leafhopper Nephotctixcincticeps ecdysone receptor ligand binding domain.

In another embodiment, the the ecdysone receptor ligand binding domainis the Christoneura fumiferana ecdysone receptor ligand binding domain,for which the amino acid sequence is set forth in SEQ ID NO: 1.

In another embodiment, the ecdysone receptor ligand binding domain is ananalog of the Christoneura fumiferana ecdysone receptor ligand bindingdomain that retains at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% or100% of the in vitro Christoneura fumiferana ecdysone receptor ligandbinding activity of the Christoneura fumiferana ecdysone receptor ligandbinding domain. In vitro ecdysone receptor ligand binding assays arewell know to those of ordinary skill in the art. For example, see WO02/066612.

In another embodiment, the ecdysone receptor ligand binding domainanalog is an ecdysone receptor ligand binding domain disclosed in WO02/066612, US 2006/0100416, WO 05/108617 and 2005/0266457. In anotherembodiment, the the ecdysone receptor ligand binding domain analog isthe V107I/Y127E substitution mutant of SEQ ID NO: 7.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for response elements. These domains may be eithernative, modified, or chimeras of different domains of heterologousreceptor proteins. The EcR, like a subset of the nuclear receptorfamily, also possesses less well-defined regions responsible forheterodimerization properties. Because the domains of nuclear receptorsare modular in nature, the LBD, DBD, and AD may be interchanged.

In another embodiment, the transcription factor comprises a AD, a DBDthat recognizes a response element associated with the therapeuticprotein or therapeutic polynucleotide whose expression is to bemodulated; and a Group H nuclear receptor LBD. In certain embodiments,the Group H nuclear receptor LBD comprises a substitution mutation.

The DNA binding domain can be any DNA binding domain (DBD) with a knownresponse element, including synthetic and chimeric DNA binding domains,or analogs, combinations, or modifications thereof. In one embodiment,the DNA binding domain is selected from the group consisting of a GAL4DBD, a LexA DBD, a transcription factor DBD, a Group H nuclear receptormember DBD, a steroid/thyroid hormone nuclear receptor superfamilymember DBD, a bacterial LacZ DBD, an EcR DBD, a GALA DBD and a LexA DBD.

The transactivation domain (abbreviated “AD” or “TA”) may be any Group Hnuclear receptor member AD, steroid/thyroid hormone nuclear receptor AD,synthetic or chimeric AD, polyglutamine AD, basic or acidic amino acidAD, a VP16 AD, a GAL4 AD, an NF-κB AD, a BP64 AD, a B42 acidicactivation domain (B42AD), a p65 transactivation domain (p65AD), or ananalog, combination, or modification thereof.

In another embodiment, the gene switch comprises a first transcriptionfactor sequence, e.g., a CAP, under the control of a first therapeuticswitch promoter (TSP-1) and a second transcription factor sequence,e.g., a LTF, under the control of a second therapeutic switch promoter(TSP-2), wherein the proteins encoded by said first transcription factorsequence and said second transcription factor sequence interact to forma protein complex (LDTFC), i.e., a “dual switch”- or “two-hybrid”-basedgene switch. The first and second TSPs may be the same or different. Inthis embodiment, the presence of two different TSPs in the gene switchthat are required for therapeutic molecule expression enhances thespecificity of the therapeutic method (see FIG. 2 of WO 2011/119773).FIG. 2 of WO 2011/119773 also demonstrates the ability to modify thetherapeutic gene switch to treat any disease, disorder, or conditionsimply by inserting the appropriate TSPs.

In a further embodiment, both the first and the second transcriptionfactor sequence, e.g., a CAP or a LTF, are under the control of a singletherapeutic switch promoter (e.g. TSP-1 in FIG. 1 of WO 2011/119773).Activation of this promoter will generate both CAP and LTF with a singleopen reading frame. This can be achieved with the use of atranscriptional linker such as an IRES (internal ribosomal entry site).In this embodiment, both portions of the ligand-dependent transcriptionfactor complex are synthesized upon activation of TSP-1. TSP-1 can be aconstitutive promoter or only activated under conditions associated withthe disease, disorder, or condition.

In a further embodiment, one transcription factor sequence, e.g. a LTF,is under the control of a therapeutic switch promoter only activatedunder conditions associated with the disease, disorder, or condition(e.g., TSP-2 or TSP-3 in FIG. 4 in WO 2011/119773) and the othertranscription factor sequence, e.g., CAP, is under the control of aconstitutive therapeutic switch promoter (e.g., TSP-1 in FIG. 4 in WO2011/119773). In this embodiment, one portion of the ligand-dependenttranscription factor complex is constitutively present while the secondportion will only be synthesized under conditions associated with thedisease, disorder, or condition.

In another embodiment, one transcription factor sequence, e.g., CAP, isunder the control of a first TSP (e.g., TSP-1 in FIG. 3 in WO2011/119773) and two or more different second transcription factorsequences, e.g., LTF-1 and LTF-2 are under the control of different TSPs(e.g., TSP-2 and TSP-3 in FIG. 3 in WO 2011/119773). In this embodiment,each of the LTFs may have a different DBD that recognizes a differentfactor-regulated promoter sequence (e.g., DBD-A binds to a responseelement associated with factor-regulated promoter-1 (FRP-1) and DBD-Bbinds to a response element associated with factor-regulated promoter-2(FRP-2). Each of the factor-regulated promoters may be operably linkedto a different therapeutic gene. In this manner, multiple treatments maybe provided simultaneously.

In one embodiment, the first transcription factor sequence encodes apolypeptide comprising a AD, a DBD that recognizes a response elementassociated with the therapeutic product sequence whose expression is tobe modulated; and a Group H nuclear receptor LBD, and the secondtranscription factor sequence encodes a transcription factor comprisinga nuclear receptor LBD selected from a vertebrate retinoid X receptor(RXR), an invertebrate RXR, an ultraspiracle protein (USP), or achimeric nuclear receptor comprising at least two different nuclearreceptor ligand binding domain polypeptide fragments selected from avertebrate RXR, an invertebrate RXR, and a USP (see WO 01/70816 A2 andUS 2004/0096942 A1). The “partner” nuclear receptor ligand bindingdomain may further comprise a truncation mutation, a deletion mutation,a substitution mutation, or another modification.

In another embodiment, the gene switch comprises a first transcriptionfactor sequence encoding a first polypeptide comprising a nuclearreceptor LBD and a DBD that recognizes a response element associatedwith the therapeutic product sequence whose expression is to bemodulated, and a second transcription factor sequence encoding a secondpolypeptide comprising an AD and a nuclear receptor LBD, wherein one ofthe nuclear receptor LBDs is a Group H nuclear receptor LBD. In oneembodiment, the first polypeptide is substantially free of an AD and thesecond polypeptide is substantially free of a DBD. For purposes of theinvention, “substantially free” means that the protein in question doesnot contain a sufficient sequence of the domain in question to provideactivation or binding activity.

In another aspect of the invention, the first transcription factorsequence encodes a protein comprising a heterodimerization partner andan AD (a “CAP”) and the second transcription factor sequence encodes aprotein comprising a DBD and a LBD (a “LTF”).

When only one nuclear receptor LBD is a Group H LBD, the other nuclearreceptor LBD may be from any other nuclear receptor that forms a dimerwith the Group H LBD. For example, when the Group H nuclear receptor LBDis an EcR LBD, the other nuclear receptor LBD “partner” may be from anEcR, a vertebrate RXR, an invertebrate RXR, an ultraspiracle protein(USP), or a chimeric nuclear receptor comprising at least two differentnuclear receptor LBD polypeptide fragments selected from a vertebrateRXR, an invertebrate RXR, or a USP (see WO 01/70816 A2, InternationalPatent Application No. PCT/US02/05235, US 2004/0096942 A1 and U.S. Pat.No. 7,531,326, incorporated herein by reference in their entirety). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

In one embodiment, the vertebrate RXR LBD is from a human Homo sapiens,mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pigSus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio,tunicate Polyandrocarpa misakiensis, or jellyfish Tripedalia cysophoraRXR.

In one embodiment, the invertebrate RXR ligand binding domain is from alocust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodidtick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an ixodid tickAmblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celucapugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog(“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphidMyzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-LepidopteranRXR homolog.

In one embodiment, the chimeric RXR LBD comprises at least twopolypeptide fragments selected from a vertebrate species RXR polypeptidefragment, an invertebrate species RXR polypeptide fragment, or anon-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment. A chimeric RXR ligand binding domain for use inthe present invention may comprise at least two different species RXRpolypeptide fragments, or when the species is the same, the two or morepolypeptide fragments may be from two or more different isoforms of thespecies RXR polypeptide fragment. Such chimeric RXR LBDs are disclosed,for example, in WO 2002/066614.

In one embodiment, the chimeric RXR ligand binding domain comprises atleast one vertebrate species RXR polypeptide fragment and oneinvertebrate species RXR polypeptide fragment.

In another embodiment, the chimeric RXR ligand binding domain comprisesat least one vertebrate species RXR polypeptide fragment and onenon-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

The ligand, when combined with the LBD of the nuclear receptor(s), whichin turn are bound to the response element of a FRP associated with atherapeutic product sequence, provides external temporal regulation ofexpression of the therapeutic product sequence. The binding mechanism orthe order in which the various components of this invention bind to eachother, that is, for example, ligand to LBD, DBD to response element, ADto promoter, etc., is not critical.

In a specific example, binding of the ligand to the LBD of a Group Hnuclear receptor and its nuclear receptor LBD partner enables expressionof the therapeutic product sequence. This mechanism does not exclude thepotential for ligand binding to the Group H nuclear receptor (GHNR) orits partner, and the resulting formation of active homodimer complexes(e.g. GHNR+GHNR or partner+partner). Preferably, one or more of thereceptor domains is varied producing a hybrid gene switch. Typically,one or more of the three domains, DBD, LBD, and AD, may be chosen from asource different than the source of the other domains so that the hybridgenes and the resulting hybrid proteins are optimized in the chosen hostcell or organism for transactivating activity, complementary binding ofthe ligand, and recognition of a specific response element. In addition,the response element itself can be modified or substituted with responseelements for other DNA binding protein domains such as the GAL-4 proteinfrom yeast (see Sadowski et al., Nature 335:563 (1988)) or LexA proteinfrom Escherichia coli (see Brent et al., Cell 43:729 (1985)), orsynthetic response elements specific for targeted interactions withproteins designed, modified, and selected for such specific interactions(see, for example, Kim et al., Proc. Natl. Acad Sci. USA, 94:3616(1997)) to accommodate hybrid receptors. Another advantage of two-hybridsystems is that they allow choice of a promoter used to drive the geneexpression according to a desired end result. Such double control may beparticularly important in areas of gene therapy, especially whencytotoxic proteins are produced, because both the timing of expressionas well as the cells wherein expression occurs may be controlled. Whengenes, operably linked to a suitable promoter, are introduced into thecells of the subject, expression of the exogenous genes is controlled bythe presence of the system of this invention. Promoters may beconstitutively or inducibly regulated or may be tissue-specific (thatis, expressed only in a particular type of cells) or specific to certaindevelopmental stages of the organism.

The DNA binding domain of the first hybrid protein binds, in thepresence or absence of a ligand, to the DNA sequence of a responseelement to initiate or suppress transcription of downstream gene(s)under the regulation of this response element.

The functional LDTFC, e.g., an EcR complex, may also include additionalprotein(s) such as immunophilins. Additional members of the nuclearreceptor family of proteins, known as transcriptional factors (such asDHR38 or betaFTZ-1), may also be ligand dependent or independentpartners for EcR, USP, and/or RXR. Additionally, other cofactors may berequired such as proteins generally known as coactivators (also termedadapters or mediators). These proteins do not bind sequence-specificallyto DNA and are not involved in basal transcription. They may exert theireffect on transcription activation through various mechanisms, includingstimulation of DNA-binding of activators, by affecting chromatinstructure, or by mediating activator-initiation complex interactions.Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as thepromiscuous coactivator C response element B binding protein, CBP/p300(for review see Glass et al., Curr. Opin. Cell Biol. 9:222 (1997)).Also, protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the unliganded EcR to silence theactivity at the response element. Current evidence suggests that thebinding of ligand changes the conformation of the receptor, whichresults in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N-CoR and SMRT (for review, see Horwitzet al., Mol Endocrinol. 10:1167 (1996)). These cofactors may either beendogenous within the cell or organism, or may be added exogenously astransgenes to be expressed in either a regulated or unregulated fashion.

B. Rapamycin Based Gene Switch

The present invention further provides a gene switch system whichutilizes FK506 binding protein as the ligand-dependent transcriptionfactor complex and rapamycin as the ligand. In one embodiment, theconstruct encoding the gene switch comprises

(a) a first polynucleotide encoding a first chimeric protein which bindsto rapamycin or an analog thereof and which comprises at least oneFK506-binding protein (FKBP) domain and at least one protein domainheterologous thereto, wherein the FKBP domain comprises a peptidesequence selected from:

-   -   (1) a naturally occurring FKBP    -   (2) a variant of a naturally occurring FKBP in which up to 10        amino acid residues have been deleted, inserted, or replaced        with substitute amino acids, and    -   (3) an FKBP encoded by a DNA sequence which selectively        hybridizes to a DNA sequence encoding an FKBP of (1) or (2);        (b) a second polynucleotide encoding a second chimeric protein        which forms a complex with both (a) rapamycin or a rapamycin        analog and (b) the first chimeric protein, and which comprises        at least one FKBP:rapamycin binding (FRB) domain and at least        one protein domain heterologous thereto, wherein the FRB domain        comprises a peptide sequence selected from:    -   (4) a naturally occurring FRB domain,    -   (5) a variant of a naturally occuring FRB domain in which up to        10 amino acid residues have been deleted, inserted, or replaced        with substitute amino acids, and    -   (6) an FRB domain encoded by a DNA sequence which selectively        hybridizes to a DNA sequence encoding an FRB of (4) or (5).

In this gene switch system, each of the first polynucleotide and thesecond polynucleotide are under the control of one or more therapeuticswitch promoters as described elsewhere herein. Furthermore, in certainembodiments, at least one protein domain heterologous to the FKBP and/orFRB domains in the first and second chimeric protein may be one or more“action” or “effector” domains. Effector domains may be selected from awide variety of protein domains including DNA binding domains,transcription activation domains, cellular localization domains andsignaling domains (i.e., domains which are capable upon clustering ormultimerization, of triggering cell growth, proliferation,differentiation, apoptosis, gene transcription, etc.).

In certain embodiments, one fusion protein contains at least one DNAbinding domain (e.g., a GAL4 or ZFHD1 DNA-binding domain) and anotherfusion protein contains at least one transcription activation domain(e.g., a VP16 or p65 transcription activation domain). Ligand-mediatedassociation of the fusion proteins represents the formation of atranscription factor complex and leads to initiation of transcription ofa target gene linked to a DNA sequence recognized by (i.e., capable ofbinding with) the DNA-binding domain on one of the fusion proteins.Information regarding the gene expression system as well as the ligandis disclosed in U.S. Pat. Nos. 6,187,757 B1, 6,649,595 B1, 6,509,152 B1,6,479,653 B1, and 6,117,680 B1.

In other embodiments, the present invention provides a gene switchsystem which comprises polynucleotides encoding two fusion proteinswhich self-aggregate in the absence of a ligand, wherein (a) the firstfusion protein comprises a conditional aggregation domain which binds toa selected ligand and a transcription activation domain, and (b) thesecond fusion protein comprising a conditional aggregation domain whichbinds to a selected ligand and a DNA binding domain, and (c) in theabsence of ligand, the cells express a gene operably linked toregulatory DNA to which said DNA binding domain binds. Modified cellscomprising the gene switch system are expanded in the presence of theligand in an amount sufficient for repression of the gene. Ligandremoval induces expression of the encoded protein that causes celldeath. The nucleic acids encoding the two fusion proteins are under thecontrol of at least one conditional promoter. The gene expression systemutilizing conditional aggregation domains is disclosed in U.S.Publication No. 2002/0048792.

C. Procaryotic Repressor/Operator Based Gene Switch System

In one embodiment, the present invention provides gene switch systemcomprising (a) a first polynucleotide coding for a transactivator fusionprotein comprising a prokaryotic tetracycline (“tet”) repressor and aeucaryotic transcriptional activator protein domain; and (b) a secondpolynucleotide coding for a therapeutic protein or therapeuticpolypeptide, wherein said second polynucleotide is operably linked to aminimal promoter and at least one tet operator sequence. The firstpolynucleotide coding for a transactivator fusion protein may comprisetherapeutic switch promoter as described elsewhere herein. Theexpression of the lethal protein is up-regulated in the absence oftetracycline. (see, e.g., Gossen et al. (1992) Proc. Natl. Acad. Sci.89: 5547-5551; Gossen et al. (1993) TIBS 18: 471-475; Furth et al.(1994) Proc. Natl. Acad. Sci. 91: 9302-9306; and Shockett et al. (1995)Proc. Natl. Acad. Sci. 92: 6522-6526). The TetO expression system isdisclosed in U.S. Pat. No. 5,464,758 B1.

In another embodiment, the gene switch system comprises the lactose(“Lac”) repressor-operator systems from the bacterium Escherichia coli.The gene switch system of the present invention may also comprise (a) afirst polynucleotide coding for a transactivator fusion proteincomprising a prokaryotic lac I repressor and a eucaryotictranscriptional activator protein domain; and (b) a secondpolynucleotide coding for a therapeutic protein or therapeuticpolypeptide, wherein said second polynucleotide is operably linked to atherapeutic switch promoter. In the Lac system, a lac operon isinactivated in the absence of lactose, or synthetic analogs such asisopropyl-b-D-thiogalactoside.

Additional gene switch systems include those described in the following:U.S. Pat. No. 7,091,038; WO2004078924; EP1266015; US20010044151;US20020110861; US20020119521; US20040033600; US20040197861;US20040235097; US20060020146; US20040049437; US20040096942;US20050228016; US20050266457; US20060100416; WO2001/70816; WO2002/29075;WO2002/066612; WO2002/066613; WO2002/066614; WO02002/066615;WO2005/108617; U.S. Pat. No. 6,258,603; US20050209283; US20050228016;US20060020146; EP0965644; U.S. Pat. No. 7,304,162; U.S. Pat. No.7,304,161; MX234742; KR10-0563143; AU765306; AU2002-248500; andAU2002-306550.

D. Combination of the Gene Switch Systems

The present invention provides nucleic acid compositions, modifiedcells, and bioreactors comprising two or more gene switch systemscomprising different ligand-dependent transcription factor complexeswhich are activated by an effective amount of one or more ligands,wherein the two or more gene switch systems comprise a first gene switchand a second gene switch, both of which selectively induce expression ofone or more therapeutic polypeptides or therapeutic polynucleotides,upon binding to one or more ligands. Within the scope of the presentinvention are any numbers of and/or combinations of gene switch systems.

In one embodiment, the present invention provides a nucleic acidcomposition comprising:

a. a first gene switch system which comprises:i. a first gene expression cassette comprising a polynucleotide encodinga first hybrid polypeptide which comprises:

-   -   1. a transactivation domain, which activates a factor-regulated        promoter operably associated with a polynucleotide encoding a        therapeutic polypeptide or therapeutic polynucleotide; and    -   2. a heterodimer partner domain,        ii. a second gene expression cassette comprising a        polynucleotide encoding a second hybrid polypeptide which        comprises:    -   1. a DNA-binding domain, which recognizes a factor-regulated        promoter operably associated with a polynucleotide encoding a        therapeutic polypeptide or therapeutic polynucleotide; and    -   2. a ligand binding domain; and        iii. a third gene expression cassette comprising a        polynucleotide encoding a therapeutic polypeptide or therapeutic        polynucleotide comprising:    -   1. a factor-regulated promoter, which is activated by the        transactivation domain of the second hybrid polypeptide; and,    -   2. a polynucleotide encoding a therapeutic polypeptide or        therapeutic polynucleotide, and        b. a second gene expression system which comprises:        i. a first gene expression cassette comprising a polynucleotide        encoding a first hybrid polypeptide which comprises:    -   1. a transactivation domain, which activates a factor-regulated        promoter operably associated with a polynucleotide encoding a        therapeutic polypeptide or therapeutic polynucleotide; and    -   2. a heterodimer partner domain,        ii. a second gene expression cassette comprising a        polynucleotide encoding a second hybrid polypeptide which        comprises:    -   1. a DNA-binding domain, which recognizes a factor-regulated        promoter operably associated with a polynucleotide encoding a        therapeutic polypeptide or therapeutic polynucleotide; and    -   2. a ligand binding domain; and        iii. a third gene expression cassette comprising a        polynucleotide encoding a therapeutic polypeptide or therapeutic        polynucleotide comprising:    -   1. a factor-regulated promoter, which is activated by the        transactivation domain of the second hybrid polypeptide; and,    -   2. a polynucleotide encoding a therapeutic polypeptide or        therapeutic polynucleotide.

The multiple inducible gene expression systems provide for expression ofa given therapeutic polynucleotide or therapeutic polypeptide underconditions associated with different diseases, disorders or conditions,or expression of multiple therapeutic polypeptides or therapeuticpolynucleotides either under the same conditions associated with thesame disease disorder or condition, or under different conditionsassociated with different diseases, disorders, or conditions.

In certain embodiments, the combination of two or more gene switchsystems may be (1) a dual-switch ecdysone receptor based gene expressionsystem and (2) a single-switch ecdysone receptor based gene switch. Inother embodiments, the combination may be (1) an single- or dual-switchecdysone receptor based gene switch and (2) a rapamycin based geneswitch. Alternatively, the combination of gene switch systems may be twoidentical rapamycin based gene switch systems disclosed above. Anypossible combinations of the gene switch systems are within the scope ofthe invention. Examples of dual-switch ecdysone systems can be found,for example, in WO 2002/29075 and US 2002/0110861.

Ligands

As used herein, the term “ligand,” as applied to LDTFC-based geneswitches e.g., EcD complex based gene switches, describes small andsoluble molecules having the capability of activating a gene switch tostimulate expression of a polypeptide encoded therein. The ligand for aligand-dependent transcription factor complex of the invention binds tothe protein complex comprising one or more of the ligand binding domain,the heterodimer partner domain, the DNA binding domain, and thetransactivation domain. The choice of ligand to activate theligand-dependent transcription factor complex depends on the type of thegene switch utilized.

Examples of ligands include, without limitation, an ecdysteroid, such asecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and thelike, 9-cis-retinoic acid, synthetic analogs of retinoic acid,N,N′-diacylhydrazines such as those disclosed in U.S. Pat. Nos.6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. PublishedApplication Nos. 2005/0209283 and 2006/0020146; oxadiazolines asdescribed in U.S. Published Application No. 2004/0171651; dibenzoylalkylcyanohydrazines such as those disclosed in European Application No.461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S.Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as thosedisclosed in European Application No. 234,994;N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.No. 4,985,461; amidoketones such as those described in U.S. PublishedApplication No. 2004/0049037; each of which is incorporated herein byreference and other similar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide,oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonateesters, juvenile hormone III, and the like. Examples of diacylhydrazineligands useful in the present invention include RG-115819(3,5-Dimethyl-benzoic acidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-methyl-3-methoxy-benzoyl)-hydrazide),RG-115932 ((R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide), andRG-115830 (3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See,e.g., U.S. patent application Ser. No. 12/155,111, published as US2009/0163592, and PCT Appl. No. PCT/US2008/006757, both of which areincorporated herein by reference in their entireties.

For example, a ligand for the edysone receptor based gene switch may beselected from any suitable ligands. Both naturally occurring ecdysone orecdyson analogs (e.g., 20-hydroxyecdysone, muristerone A, ponasterone A,ponasterone B, ponasterone C, 26-iodoponasterone A, inokosterone or26-mesylinokosterone) and non-steroid inducers may be used as a ligandfor gene switch of the present invention. U.S. Pat. No. 6,379,945 B1,describes an insect steroid receptor isolated from Heliothis virescens(“HEcR”) which is capable of acting as a gene switch responsive to bothsteroid and certain non-steroidal inducers. Non-steroidal inducers havea distinct advantage over steroids, in this and many other systems whichare responsive to both steroids and non-steroid inducers, for a numberof reasons including, for example: lower manufacturing cost, metabolicstability, absence from insects, plants, or mammals, and environmentalacceptability. U.S. Pat. No. 6,379,945 BI describes the utility of twodibenzoylhydrazines, 1,2-dibenzoyl-1-tert-butyl-hydrazine andtebufenozide(N-(4-ethylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butyl-hydrazine) asligands for an ecdysone-based gene switch. Also included in the presentinvention as a ligand are other dibenzoylhydrazines, such as thosedisclosed in U.S. Pat. No. 5,117,057 B1. Use of tebufenozide as achemical ligand for the ecdysone receptor from Drosophila melanogasteris also disclosed in U.S. Pat. No. 6,147,282. Additional, non-limitingexamples of ecdysone ligands are3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, a1,2-diacyl hydrazine, an N′-substituted-N,N′-disubstituted hydrazine, adibenzoylalkyl cyanohydrazine, an N-substituted-N-alkyl-N,N-diaroylhydrazine, an N-substituted-N-acyl-N-alkyl, carbonyl hydrazine or anN-aroyl-N′-alkyl-N′-aroyl hydrazine. (See U.S. Pat. No. 6,723,531).

In one embodiment, the ligand for an ecdysone based gene switch systemis a diacylhydrazine ligand or chiral diacylhydrazine ligand. The ligandused in the gene switch system may be compounds of Formula I

whereinA is alkoxy, arylalkyloxy or aryloxy;B is optionally substituted aryl or optionally substituted heteroaryl;andR¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclo, optionally substituted aryl or optionallysubstituted heteroaryl;or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In another embodiment, the ligand may be enantiomerically enrichedcompounds of Formula II

whereinA is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;B is optionally substituted aryl or optionally substituted heteroaryl;andR¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclo, optionally substituted aryl or optionallysubstituted heteroaryl;with the proviso that R¹ does not equal R²;wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly S;or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In certain embodiments, the ligand may be enantiomerically enrichedcompounds of Formula III

whereinA is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substitutedaryl or optionally substituted heteroaryl;B is optionally substituted aryl or optionally substituted heteroaryl;andR¹ and R² are independently optionally substituted alkyl, arylalkyl,hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclo, optionally substituted aryl or optionallysubstituted heteroaryl;with the proviso that R¹ does not equal R²;wherein the absolute configuration at the asymmetric carbon atom bearingR¹ and R² is predominantly R;or pharmaceutically acceptable salts, hydrates, crystalline forms oramorphous forms thereof.

In one embodiment, a ligand may be (R)-3,5-dimethyl-bcnzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide havingan enantiomeric excess of at least 95% or a pharmaceutically acceptablesalt, hydrate, crystalline form or amorphous form thereof.

The diacylhydrazine ligands of Formula I and chiral diacylhydrazineligands of Formula II or III, when used with an ecdysone-based geneswitch system, provide the means for external temporal regulation ofexpression of a therapeutic polypeptide or therapeutic polynucleotide ofthe present invention. See U.S. application Ser. No. 12/155,111,published as US 2009/0163592, filed May 29, 2008, which is fullyincorporated by reference herein.

The ligands used in the present invention may form salts. The term“salt(s)” as used herein denotes acidic and/or basic salts formed withinorganic and/or organic acids and bases. In addition, when a compoundof Formula I, II or III contains both a basic moiety and an acidicmoiety, zwitterions (“inner salts”) may be formed and are includedwithin the term “salt(s)” as used herein. Pharmaceutically acceptable(i.e., non-toxic, physiologically acceptable) salts are used, althoughother salts are also useful, e.g., in isolation or purification stepswhich may be employed during preparation. Salts of the compounds ofFormula I, II or III may be formed, for example, by reacting a compoundwith an amount of acid or base, such as an equivalent amount, in amedium such as one in which the salt precipitates or in an aqueousmedium followed by lyophilization.

The ligands which contain a basic moiety may form salts with a varietyof organic and inorganic acids. Exemplary acid addition salts includeacetates (such as those formed with acetic acid or trihaloacetic acid,for example, trifluoroacetic acid), adipates, alginates, ascorbates,aspartates, benzoates, benzenesulfonates, bisulfates, borates,butyrates, citrates, camphorates, camphorsulfonates,cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides (formed withhydrochloric acid), hydrobromides (formed with hydrogen bromide),hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed withmaleic acid), methanesulfonates (formed with methanesulfonic acid),2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates,persulfates, 3-phenylpropionates, phosphates, picrates, pivalates,propionates, salicylates, succinates, sulfates (such as those formedwith sulfuric acid), sulfonates (such as those mentioned herein),tartrates, thiocyanates, toluenesulfonates such as tosylates,undecanoates, and the like.

The ligands which contain an acidic moiety may form salts with a varietyof organic and inorganic bases. Exemplary basic salts include ammoniumsalts, alkali metal salts such as sodium, lithium, and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases (for example, organic amines) such as benzathines,dicyclohexylamines, hydrabamines (formed withN,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine and the like.

Non-limiting examples of the ligands for the inducible gene expressionsystem utilizing the FK506 binding domain are FK506, Cyclosporin A, orRapamycin. FK506, rapamycin, and their analogs are disclosed in U.S.Pat. Nos. 6,649,595 B2 and 6,187,757. See also U.S. Pat. Nos. 7,276,498and 7,273,874.

The ligands described herein may be administered alone or as part of apharmaceutical composition comprising a pharmaceutically acceptablecarrier. In one embodiment, the pharmaceutical composition are in theform of solutions, suspensions, tablets, capsules, ointments, elixirs,or injectable compositions.

In one embodiment, the polynucleotide encoding an antibody encodes amonoclonal antibody.

In another embodiment, the vector and methods of the present inventioncan be used to express nucleic acid as a vaccine. The present inventionalso provides a vaccine composition comprising a vector or expressionsystem of the present invention. In another embodiment, the vaccinecomposition comprises an adjuvant.

An “erythropoietin or agonist thereof” is an erythropoietin polypeptide,a polypeptide having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity with an erythropoietin, or a fragment of anerythropoietin that retains at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% of the in vitro erythropoietin receptor bindingactivity of erythropoietin.

Any erythropoictin polynucleotide sequence can be used in the method ofthe present invention. In one embodiment, the erythropoietinpolynucleotide sequence encodes human erythropoietin, for which theamino acid sequence is set forth in Accession No. AAF23134 (SEQ ID NO:6). The amino acid sequences coding for erythropoietin are alsoavailable from public databases as accession numbers AAH93628 (human);AA19266 (mouse); and BAA01593 (rat), sequences of which are incorporatedby reference herein. The polynucleotide sequences coding forerythropoietin are available from public databases as accession numbersBC093628 (human); BC119265 (mouse); and D10763 (rat), sequences of whichare incorporated by reference herein.

In another embodiment, the erythropoietin polynucleotide sequenceencodes is analog of human erythropoietin that retains at least 80%,85%, 90%, 95%, 96%, 97%, 98% 99% or 100% of the in vitro erythropoietinreceptor binding activity of human erythropoietin. In vitroerythropoietin receptor binding assays are well know to those ofordinary skill in the art. For example, see Harris, K. W. et al., J.Biol. Chem. 25: 15205-9 (1992); Wrighton, N. C. et al., Science 273:458-463 (1996); and Jarsch, M. et al., Pharmacology 81: 63-69 (2008).

Non-limiting examples of erthropoietins include darbepoietin alfa,epoetin alfa, epoetin alfa, epoetin beta, and epoetin kappa.

Non-limiting examples of ertythropoietin agonists include those agonistsdisclosed in U.S. Pat. Nos. 7,767,643; 7,786,163; 7,674,913; 7,553,861;7,410,941; 7,345,019; 7,309,687; 6,531,121; 5,858,670; 5,650,489; and5,510,240. Other non-limiting examples of ertythropoietin agonistsinclude those agonists disclosed in in U.S. patent publication nos.2011/0027890, 2010/0305002, 2010/297117, 2010/0297106, 2010/0190692,2010/0145006, 2010/136015, 2010/0120661, 2010/093608, 2010/0028331,2010/016218, 2010/0009961, 2009/0233844, 2009/0022734, 2009/0004202,2008/0213277, 2008/0014193, 2007/0298031, 2007/0293421, 2007/0060547,2006/027071, 2006/0009518, 2003/0134798, 2003/0104988 and 2002/008616.Other non-limiting examples of ertythropoietin agonists include thoseagonists disclosed in MacDougal, I. C. et al., N. Engl. J. Med. 361:1848-55 (2009); Pankratova, S. et al., Brain 133(Pt. 8): 2281-94 (2010);and Zarychanski, R. et al., Canadian Medical Association Journal 177:725-34 (2007).

The term “ecdysone receptor-based,” with respect to a gene switch,refers to a gene switch comprising at least a functional part of anaturally occurring or synthetic ecdysone receptor ligand binding domainand which regulates gene expression in response to a ligand that bindsto the ecdysone receptor ligand binding domain. Examples ofecdysone-responsive systems are described in U.S. Pat. Nos. 7,091,038and 6,258,603. In one embodiment, the system is the RheoSwitch®Therapeutic System (RTS), which contains two fusion proteins, the DEFdomains of a mutagenized ecdysone receptor (EcR) fused with a Gal4 DNAbinding domain and the EF domains of a chimeric RXR fused with a VP16transcription activation domain, expressed under a constitutive promoteras illustrated in FIG. 1.

The terms “modulate” and “modulates” mean to induce, reduce or inhibitnucleic acid or gene expression, resulting in the respective induction,reduction or inhibition of protein or polypeptide production.

The polynucleotides or vectors according to the invention may furthercomprise at least one promoter suitable for driving expression of a genein a host cell.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor 1 (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may also be derived from various genes native to thepreferred hosts. Optionally, a termination site may be unnecessary,however, it is most preferred if included. In one embodiment of theinvention, the termination control region may be comprised or be derivedfrom a synthetic sequence, synthetic polyadenylation signal, an SV40late polyadenylation signal, an SV40 polyadenylation signal, a bovinegrowth hormone (BGH) polyadenylation signal, viral terminator sequences,or the like.

The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)”refer to DNA sequences located downstream (3′) of a coding sequence andmay comprise polyadenylation [poly(A)] recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor.

“Regulatory region” refers to a nucleic acid sequence that regulates theexpression of a second nucleic acid sequence. A regulatory region mayinclude sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin that are responsible for expressing differentproteins or even synthetic proteins (a heterologous region). Inparticular, the sequences can be sequences of prokaryotic, eukaryotic,or viral genes or derived sequences that stimulate or represstranscription of a gene in a specific or non-specific manner and in aninducible or non-inducible manner. Regulatory regions include origins ofreplication, RNA splice sites, promoters, enhancers, transcriptionaltermination sequences, and signal sequences which direct the polypeptideinto the secretory pathways of the target cell.

A regulatory region from a “heterologous source” refers to a regulatoryregion that is not naturally associated with the expressed nucleic acid.Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene. The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or thecoding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA,or other RNA that is not translated yet has an effect on cellularprocesses.

“Polypeptide,” “peptide” and “protein” are used interchangeably andrefer to a polymeric compound comprised of covalently linked amino acidresidues.

An “isolated polypeptide,” “isolated peptide” or “isolated protein”refer to a polypeptide or protein that is substantially free of thosecompounds that are normally associated therewith in its natural state(e.g., other proteins or polypeptides, nucleic acids, carbohydrates,lipids). “Isolated” is not meant to exclude artificial or syntheticmixtures with other compounds, or the presence of impurities which donot interfere with biological activity, and which may be, for example,due to incomplete purification, addition of stabilizers, or compoundinginto a pharmaceutically acceptable preparation.

A “substitution mutant polypeptide” or a “substitution mutant” will beunderstood to mean a mutant polypeptide comprising a substitution of atleast one wild-type or naturally occurring amino acid with a differentamino acid relative to the wild-type or naturally occurring polypeptide.A substitution mutant polypeptide may comprise only one wild-type ornaturally occurring amino acid substitution and may be referred to as a“point mutant” or a “single point mutant” polypeptide. Alternatively, asubstitution mutant polypeptide may comprise a substitution of two ormore wild-type or naturally occurring amino acids with two or more aminoacids relative to the wild-type or naturally occurring polypeptide.According to the invention, a Group H nuclear receptor ligand bindingdomain polypeptide comprising a substitution mutation comprises asubstitution of at least one wild-type or naturally occurring amino acidwith a different amino acid relative to the wild-type or naturallyoccurring Group H nuclear receptor ligand binding domain polypeptide.Non-limiting examples of substitution mutant Group H nuclear receptorligand binding domain polypeptides are found in WO 2002/066612 and US2006/0100416.

When the substitution mutant polypeptide comprises a substitution of twoor more wild-type or naturally occurring amino acids, this substitutionmay comprise either an equivalent number of wild-type or naturallyoccurring amino acids deleted for the substitution, i.e., 2 wild-type ornaturally occurring amino acids replaced with 2 non-wild-type ornon-naturally occurring amino acids, or a non-equivalent number ofwild-type amino acids deleted for the substitution, i.e., 2 wild-typeamino acids replaced with 1 non-wild-type amino acid (asubstitution+deletion mutation), or 2 wild-type amino acids replacedwith 3 non-wild-type amino acids (a substitution+insertion mutation).

Substitution mutants may be described using an abbreviated nomenclaturesystem to indicate the amino acid residue and number replaced within thereference polypeptide sequence and the new substituted amino acidresidue. For example, a substitution mutant in which the twentieth(20^(th)) amino acid residue of a polypeptide is substituted may beabbreviated as “x20z”, wherein “x” is the amino acid to be replaced,“20” is the amino acid residue position or number within thepolypeptide, and “z” is the new substituted amino acid. Therefore, asubstitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala”indicates that the mutant comprises an alanine residue (commonlyabbreviated in the art as “A” or “Ala”) in place of the glutamic acid(commonly abbreviated in the art as “E” or “Glu”) at position 20 of thepolypeptide.

A substitution mutation may be made by any technique for mutagenesisknown in the art, including but not limited to, in vitro site-directedmutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978); Zolleret al., DNA 3:479 (1984); Oliphant et al., Gene 44:177 (1986);Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986)), use ofTAB® linkers (Pharmacia), restriction endonuclease digestion/fragmentdeletion and substitution, PCR-mediated/oligonucleotide-directedmutagenesis, and the like. PCR-based techniques are preferred forsite-directed mutagenesis (see Higuchi, 1989, “Using PCR to EngineerDNA”, in PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

The term “fragment,” as applied to a polypeptide, refers to apolypeptide whose amino acid sequence is shorter than that of thereference polypeptide and which comprises, over the entire portion withthese reference polypeptides, an identical amino acid sequence. Suchfragments may, where appropriate, be included in a larger polypeptide ofwhich they are a part. Such fragments of a polypeptide according to theinvention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200,240, or 300 or more amino acids.

A “variant” of a polypeptide or protein refers to any analogue,fragment, derivative, or mutant which is derived from a polypeptide orprotein and which retains at least one biological property of thepolypeptide or protein. Different variants of the polypeptide or proteinmay exist in nature. These variants may be allelic variationscharacterized by differences in the nucleotide sequences of thestructural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canproduce variants having single or multiple amino acid substitutions,deletions, additions, or replacements. These variants may include, interalia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids, (b)variants in which one or more amino acids are added to the polypeptideor protein, (c) variants in which one or more of the amino acidsincludes a substituent group, and (d) variants in which the polypeptideor protein is fused with another polypeptide such as serum albumin. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art. Inone embodiment, a variant polypeptide comprises at least about 14 aminoacids.

The term “homology” refers to the percent of identity between twopolynucleotide or two polypeptide moieties. The correspondence betweenthe sequence from one moiety to another can be determined by techniquesknown to the art. For example, homology can be determined by a directcomparison of the sequence information between two polypeptide moleculesby aligning the sequence information and using readily availablecomputer programs. Alternatively, homology can be determined byhybridization of polynucleotides under conditions that form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s) and size determination of thedigested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667 (1987)). Such proteins (and their encoding genes)have sequence homology, as reflected by their high degree of sequencesimilarity. However, in common usage and in the application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and not a common evolutionary origin.

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that may or may not share a commonevolutionary origin (see Reeck et al., Cell 50:667 (1987)). In oneembodiment, two DNA sequences are “substantially homologous” or“substantially similar” when at least about 50% (e.g., at least about75%, 90%, or 95%) of the nucleotides match over the defined length ofthe DNA sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data banks, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart (see e.g., Sambrook et al., 1989, supra).

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. “Substantially similar” alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate alteration of gene expression by antisense or co-suppressiontechnology. “Substantially similar” also refers to modifications of thenucleic acid fragments of the invention such as deletion or insertion ofone or more nucleotide bases that do not substantially affect thefunctional properties of the resulting transcript. It is thereforeunderstood that the invention encompasses more than the specificexemplary sequences. Each of the proposed modifications is well withinthe routine skill in the art, as is determination of retention ofbiological activity of the encoded products.

Two amino acid sequences are “substantially homologous” or“substantially similar” when greater than about 40% of the amino acidsare identical, or greater than 60% are similar (functionally identical).Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

A “substantial portion” of an amino acid or nucleotide sequencecomprises enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al., J. Mol. Biol. 215:403 (1993)); available atncbi.nlm.nih.gov/BLAST/). In general, a sequence often or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence.

The term “percent identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, New York (1991). Preferredmethods to determine identity are designed to give the best matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing sequence analysis software such as the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences may be performed using the Clustalmethod of alignment (Higgins et al., CABIOS. 5:151 (1989)) with thedefault parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method may beselected: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwareincludes, but is not limited to, the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.),BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)),and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA).Within the context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters which originally load with thesoftware when first initialized.

“Chemically synthesized,” as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

As used herein, two or more individually operable gene regulationsystems are said to be “orthogonal” when; a) modulation of each of thegiven systems by its respective ligand, at a chosen concentration,results in a measurable change in the magnitude of expression of thegene of that system, and b) the change is statistically significantlydifferent than the change in expression of all other systemssimultaneously operable in the cell, tissue, or organism, regardless ofthe simultaneity or sequentiality of the actual modulation. Preferably,modulation of each individually operable gene regulation system effectsa change in gene expression at least 2-fold greater than all otheroperable systems in the cell, tissue, or organism, e.g., at least5-fold, 10-fold, 100-fold, or 500-fold greater. Ideally, modulation ofeach of the given systems by its respective ligand at a chosenconcentration results in a measurable change in the magnitude ofexpression of the gene of that system and no measurable change inexpression of all other systems operable in the cell, tissue, ororganism. In such cases the multiple inducible gene regulation system issaid to be “fully orthogonal.” Useful orthogonal ligands and orthogonalreceptor-based gene expression systems are described in US 2002/0110861A1.

The term “exogenous gene” means a gene foreign to the subject, that is,a gene which is introduced into the subject through a transformationprocess, an unmutated version of an endogenous mutated gene or a mutatedversion of an endogenous unmutated gene. The method of transformation isnot critical to this invention and may be any method suitable for thesubject known to those in the art. Exogenous genes can be either naturalor synthetic genes which are introduced into the subject in the form ofDNA or RNA which may function through a DNA intermediate such as byreverse transcriptase. Such genes can be introduced into target cells,directly introduced into the subject, or indirectly introduced by thetransfer of transformed cells into the subject.

The term “therapeutic product” refers to a therapeutic polypeptide ortherapeutic polynucleotide which imparts a beneficial function to thehost cell in which such product is expressed. Therapeutic polypeptidesmay include, without limitation, peptides as small as three amino acidsin length, single- or multiple-chain proteins, and fusion proteins.Therapeutic polynucleotides may include, without limitation, antisenseoligonucleotides, small interfering RNAs, ribozymes, and RNA externalguide sequences. The therapeutic product may comprise a naturallyoccurring sequence, a synthetic sequence or a combination of natural andsynthetic sequences.

The term “ligand-dependent transcription factor complex” or “LDTFC”refers to a transcription factor comprising one or more proteinsubunits, which complex can regulate gene expression driven by a“factor-regulated promoter” as defined herein. A model LDTFC is an“ecdysone receptor complex” generally refers to a heterodimeric proteincomplex having at least two members of the nuclear receptor family,ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins (see Yao etal., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)). Afunctional LDTFC such as an EcR complex may also include additionalprotein(s) such as immunophilins. Additional members of the nuclearreceptor family of proteins, known as transcriptional factors (such asDHR38, betaFTZ-1 or other insect homologs), may also be ligand dependentor independent partners for EcR and/or USP. A LDTFC such as an EcRcomplex can also be a heterodimer of EcR protein and the vertebratehomolog of ultraspiracle protein, retinoic acid-X-receptor (“RXR”)protein or a chimera of USP and RXR. The terms “LDTFC” and “EcR complex”also encompass homodimer complexes of the EcR protein or USP, as well assingle polypeptides or trimers, tetramer, and other multimers servingthe same function.

A LDTFC such as an EcR complex can be activated by an active ecdysteroidor non-steroidal ligand bound to one of the proteins of the complex,inclusive of EcR, but not excluding other proteins of the complex. ALDTFC such as an EcR complex includes proteins which are members of thenuclear receptor superfamily wherein all members are characterized bythe presence of one or more polypeptide subunits comprising anamino-terminal transactivation domain (“AD,” “TD,” or “TA,” usedinterchangeably herein), a DNA binding domain (“DBD”), and a ligandbinding domain (“LBD”). The AD may be present as a fusion with a“heterodimerization partner” or “HP.” A fusion protein comprising an ADand HP of the invention is referred to herein as a “coactivationprotein” or “CAP.” The DBD and LBD may be expressed as a fusion protein,referred to herein as a “ligand-inducible transcription factor (“LTF”).The fusion partners may be separated by a linker, e.g., a hinge region.Some members of the LTF family may also have another transactivationdomain on the carboxy-terminal side of the LBD. The DBD is characterizedby the presence of two cysteine zinc fingers between which are two aminoacid motifs, the P-box and the D-box, which confer specificity forecdysone response elements. These domains may be either native,modified, or chimeras of different domains of heterologous receptorproteins.

The DNA sequences making up the exogenous gene, the response element,and the LDTFC, e.g., EcR complex, may be incorporated intoarchaebacteria, procaryotic cells such as Escherichia coli, Bacillussubtilis, or other enterobacteria, or eucaryotic cells such as plant oranimal cells. However, because many of the proteins expressed by thegene are processed incorrectly in bacteria, eucaryotic cells arepreferred. The cells may be in the form of single cells or multicellularorganisms. The nucleotide sequences for the exogenous gene, the responseelement, and the receptor complex can also be incorporated as RNAmolecules, preferably in the form of functional viral RNAs such astobacco mosaic virus. Of the eucaryotic cells, vertebrate cells arepreferred because they naturally lack the molecules which conferresponses to the ligands of this invention for the EcR. As a result,they are “substantially insensitive” to the ligands of this invention.Thus, the ligands useful in this invention will have negligiblephysiological or other effects on transformed cells, or the wholeorganism. Therefore, cells can grow and express the desired product,substantially unaffected by the presence of the ligand itself.

The term “ecdysone receptor complex” generally refers to a heterodimericprotein complex having at least two members of the nuclear receptorfamily, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins(see Yao et al., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)).The functional EcR complex may also include additional protein(s) suchas immunophilins. Additional members of the nuclear receptor family ofproteins, known as transcriptional factors (such as DHR38, betaFTZ-1 orother insect homologs), may also be ligand dependent or independentpartners for EcR and/or USP. The EcR complex can also be a heterodimerof EcR protein and the vertebrate homolog of ultraspiracle protein,retinoic acid-X-receptor (“RXR”) protein or a chimera of USP and RXR.The term EcR complex also encompasses homodimer complexes of the EcRprotein or USP.

An EcR complex can be activated by an active ecdysteroid ornon-steroidal ligand bound to one of the proteins of the complex,inclusive of EcR, but not excluding other proteins of the complex. Asused herein, the term “ligand,” as applied to EcR-based gene switches,describes small and soluble molecules having the capability ofactivating a gene switch to stimulate expression of a polypeptideencoded therein. Examples of ligands include, without limitation, anecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A,muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs ofretinoic acid, N,N′-diacylhydrazines such as those disclosed in U.S.Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S.Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolinesas described in U.S. Published Application No. 2004/0171651;dibenzoylalkyl cyanohydrazines such as those disclosed in EuropeanApplication No. 461,809; N-alkyl-N,N′-diaroylhydrazines such as thosedisclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazinessuch as those disclosed in European Application No. 234,994;N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat.No. 4,985,461; amidoketones such as those described in U.S. PublishedApplication No. 2004/0049037; and other similar materials including3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide,oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol,25-epoxycholesterol, T0901317,5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonateesters, juvenile hormone III, and the like. Examples of diacylhydrazineligands useful in the invention include RG-115819 (3,5-Dimethyl-benzoicacidN-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-methyl-3-methoxy-benzoyl)-hydrazide),RG-115932 ((R)-3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide), andRG-115830 (3,5-Dimethyl-benzoic acidN-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide). SeeU.S. application Ser. No. 12/155,111, filed May 29, 2008, andPCT/US2008/006757 filed May 29, 2008, for additional diacylhydrazinesthat are useful in the practice of the invention.

The EcR complex includes proteins which are members of the nuclearreceptor superfamily wherein all members are characterized by thepresence of an amino-terminal transactivation domain (“TA”), a DNAbinding domain (“DBD”), and a ligand binding domain (“LBD”) separated bya hinge region. Some members of the family may also have anothertransactivation domain on the carboxy-terminal side of the LBD. The DBDis characterized by the presence of two cysteine zinc fingers betweenwhich are two amino acid motifs, the P-box and the D-box, which conferspecificity for ecdysone response elements. These domains may be eithernative, modified, or chimeras of different domains of heterologousreceptor proteins.

The DNA sequences making up the exogenous gene, the response element,and the EcR complex may be incorporated into archaebacteria, procaryoticcells such as Escherichia coli, Bacillus subtilis, or otherenterobacteria, or eucaryotic cells such as plant or animal cells.However, because many of the proteins expressed by the gene areprocessed incorrectly in bacteria, eucaryotic cells are preferred. Thecells may be in the form of single cells or multicellular organisms. Thenucleotide sequences for the exogenous gene, the response element, andthe receptor complex can also be incorporated as RNA molecules,preferably in the form of functional viral RNAs such as tobacco mosaicvirus. Of the eucaryotic cells, vertebrate cells are preferred becausethey naturally lack the molecules which confer responses to the ligandsof this invention for the EcR. As a result, they are “substantiallyinsensitive” to the ligands of this invention. Thus, the ligands usefulin this invention will have negligible physiological or other effects ontransformed cells, or the whole organism. Therefore, cells can grow andexpress the desired product, substantially unaffected by the presence ofthe ligand itself.

EcR ligands, when used with the EcR complex which in turn is bound tothe response element linked to an exogenous gene provide the means forexternal temporal regulation of expression of the exogenous gene. Theorder in which the various components bind to each other, that is,ligand to receptor complex and receptor complex to response element, isnot critical. Typically, modulation of expression of the exogenous geneis in response to the binding of the EcR complex to a specific control,or regulatory, DNA element. The EcR protein, like other members of thenuclear receptor family, possesses at least three domains, atransactivation domain, a DNA binding domain, and a ligand bindingdomain. This receptor, like a subset of the nuclear receptor family,also possesses less well-defined regions responsible forheterodimerization properties. Binding of the ligand to the ligandbinding domain of EcR protein, after heterodimerization with USP or RXRprotein, enables the DNA binding domains of the heterodimeric proteinsto bind to the response element in an activated form, thus resulting inexpression or suppression of the exogenous gene. This mechanism does notexclude the potential for ligand binding to either EcR or USP, and theresulting formation of active homodimer complexes (e.g., EcR+EcR orUSP+USP). In one embodiment, one or more of the receptor domains can bevaried producing a chimeric gene switch. Typically, one or more of thethree domains may be chosen from a source different than the source ofthe other domains so that the chimeric receptor is optimized in thechosen host cell or organism for transactivating activity, complementarybinding of the ligand, and recognition of a specific response element.In addition, the response element itself can be modified or substitutedwith response elements for other DNA binding protein domains such as theGAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988) orLexA protein from E. coli (see Brent et al., Cell 43:729 (1985)) toaccommodate chimeric EcR complexes. Another advantage of chimericsystems is that they allow choice of a promoter used to drive theexogenous gene according to a desired end result. Such double controlcan be particularly important in areas of gene therapy, especially whencytotoxic proteins are produced, because both the timing of expressionas well as the cells wherein expression occurs can be controlled. Whenexogenous genes, operatively linked to a suitable promoter, areintroduced into the cells of the subject, expression of the exogenousgenes is controlled by the presence of the ligand of this invention.Promoters may be constitutively or inducibly regulated or may betissue-specific (that is, expressed only in a particular type of cell)or specific to certain developmental stages of the organism.

In certain embodiments, the therapeutic switch promoter described in themethods is consititutive. In certain embodiments, the therapeutic switchpromoter is activated under conditions associated with a disease,disorder, or condition, e.g., the promoter is activated in response to adisease, in response to a particular physiological, developmental,differentiation, or pathological condition, and/or in response to one ormore specific biological molecules; and/or the promoter is activated inparticular tissue or cell types. In certain embodiments, the disease,disorder, or condition is responsive to the therapeutic polypeptide orpolynucleotide. For example in certain non-limiting embodiments thetherapeutic polynucleotide or polypeptide is useful to treat, prevent,ameliorate, reduce symptoms, prevent progression, or cure the disease,disorder or condition, but need not accomplish any one or all of thesethings. In certain embodiments, the first and second polynucleotides areintroduced so as to permit expression of the ligand-dependenttranscription factor complex under consitions associated with a disease,disorder or condition. In one embodiment, the therapeutic methods arecarried out such that the therapeutic polypeptide or therapeuticpolynucleotide is expressed and disseminated through the subject at alevel sufficient to treat, ameliorate, or prevent said disease,disorder, or condition. As used herein, “disseminated” means that thepolypeptide is expressed and released from the modified cellsufficiently to have an effect or activity in the subject. Disseminationmay be systemic, local or anything in between. For example, thetherapeutic polypeptide or therapeutic polynucleotide might besystemically disseminated through the bloodstream or lymph system.Alternatively, the therapeutic polypeptide or therapeutic polynucleotidemight be disseminated locally in a tissue or organ to be treated.

Numerous genomic and cDNA nucleic acid sequences coding for a variety ofpolypeptides, such as transcription factors and reporter proteins, arewell known in the art. Those skilled in the art have access to nucleicacid sequence information for virtually all known genes and can eitherobtain the nucleic acid molecule directly from a public depository, theinstitution that published the sequence, or employ routine methods toprepare the molecule. See for example the description of the sequenceaccession numbers, infra.

The gene switch may be any gene switch system that regulates geneexpression by addition or removal of a specific ligand. In oneembodiment, the gene switch is one in which the level of gene expressionis dependent on the level of ligand that is present. Examples ofligand-dependent transcription factors that may be used in the geneswitches of the invention include, without limitation, members of thenuclear receptor superfamily activated by their respective ligands(e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, andanalogs and mimetics thereof) and rTTA activated by tetracycline. In oneaspect of the invention, the gene switch is an EcR-based gene switch.Examples of such systems include, without limitation, the systemsdescribed in U.S. Pat. Nos. 6,258,603, 7,045,315, U.S. Published PatentApplication Nos. 2006/0014711, 2007/0161086, and International PublishedApplication No. WO 01/70816. Examples of chimeric ecdysone receptorsystems are described in U.S. Pat. No. 7,091,038, U.S. Published PatentApplication Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457,and 2006/0100416, and International Published Application Nos. WO01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO02/29075, and WO 2005/108617. An example of a non-steroidal ecdysoneagonist-regulated system is the RheoSwitch® Mammalian InducibleExpression System (New England Biolabs, Ipswich, Mass.).

In one embodiment, a polynucleotide encoding the gene switch comprises asingle transcription factor sequence encoding a ligand-dependenttranscription factor under the control of a promoter. The transcriptionfactor sequence may encode a ligand-dependent transcription factor thatis a naturally occurring or an artificial transcription factor. Anartificial transcription factor is one in which the natural sequence ofthe transcription factor has been altered, e.g., by mutation of thesequence or by the combining of domains from different transcriptionfactors. In one embodiment, the transcription factor comprises a Group Hnuclear receptor ligand binding domain (LBD). In one embodiment, theGroup H nuclear receptor LBD is from an EcR, a ubiquitous receptor, anorphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, aretinoid X receptor interacting protein-15, a liver X receptor β, asteroid hormone receptor like protein, a liver X receptor, a liver Xreceptor α, a farnesoid X receptor, a receptor interacting protein 14,or a farnesol receptor. In another embodiment, the Group H nuclearreceptor LBD is from an ecdysone receptor.

In one embodiment, a polynucleotide encoding the gene switch comprises asingle transcription factor sequence encoding a ligand-dependenttranscription factor under the control of a promoter. The transcriptionfactor sequence may encode a ligand-dependent transcription factor thatis a naturally occurring or an artificial transcription factor. Anartificial transcription factor is one in which the natural sequence ofthe transcription factor has been altered, e.g., by mutation of thesequence or by the combining of domains from different transcriptionfactors. In one embodiment, the transcription factor comprises a Group Hnuclear receptor ligand binding domain (LBD). In one embodiment, theGroup H nuclear receptor LBD is from an EcR, a ubiquitous receptor, anorphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, aretinoid X receptor interacting protein-15, a liver X receptor β, asteroid hormone receptor like protein, a liver X receptor, a liver Xreceptor α, a farnesoid X receptor, a receptor interacting protein 14,or a farnesol receptor. In another embodiment, the Group H nuclearreceptor LBD is from an ecdysone receptor.

The EcR and the other Group H nuclear receptors are members of thenuclear receptor superfamily wherein all members are generallycharacterized by the presence of an amino-terminal transactivationdomain (TD), a DNA binding domain (DBD), and a LBD separated from theDBD by a hinge region. As used herein, the term “DNA binding domain”comprises a minimal polypeptide sequence of a DNA binding protein, up tothe entire length of a DNA binding protein, so long as the DNA bindingdomain functions to associate with a particular response element.Members of the nuclear receptor superfamily are also characterized bythe presence of four or five domains: A/B, C, D, E, and in some membersF (see U.S. Pat. No. 4,981,784 and Evans, Science 240:889 (1988)). The“A/B” domain corresponds to the transactivation domain, “C” correspondsto the DNA binding domain, “D” corresponds to the hinge region, and “E”corresponds to the ligand binding domain. Some members of the family mayalso have another transactivation domain on the carboxy-terminal side ofthe LBD corresponding to “F”.

The DBD is characterized by the presence of two cysteine zinc fingersbetween which are two amino acid motifs, the P-box and the D-box, whichconfer specificity for response elements. These domains may be eithernative, modified, or chimeras of different domains of heterologousreceptor proteins. The EcR, like a subset of the nuclear receptorfamily, also possesses less well-defined regions responsible forheterodimerization properties. Because the domains of nuclear receptorsare modular in nature, the LBD, DBD, and TD may be interchanged.

In another embodiment, the transcription factor comprises a TD, a DBDthat recognizes a response element associated with the exogenous genewhose expression is to be modulated; and a Group H nuclear receptor LBD.In certain embodiments, the Group H nuclear receptor LBD comprises asubstitution mutation.

In another embodiment, a polynucleotide encoding the gene switchcomprises a first transcription factor sequence under the control of afirst promoter and a second transcription factor sequence under thecontrol of a second promoter, wherein the proteins encoded by said firsttranscription factor sequence and said second transcription factorsequence interact to form a protein complex which functions as aligand-dependent transcription factor, i.e., a “dual switch”- or“two-hybrid”-based gene switch. The first and second promoters may bethe same or different.

In certain embodiments, the polynucleotide encoding a gene switchcomprises a first transcription factor sequence and a secondtranscription factor sequence under the control of a promoter, whereinthe proteins encoded by said first transcription factor sequence andsaid second transcription factor sequence interact to form a proteincomplex which functions as a ligand-dependent transcription factor,i.e., a “single gene switch”. The first transcription factor sequenceand a second transcription factor sequence may be connected by aninternal ribosomal entry site (IRES). The IRES may be an EMCV IRES.

In one embodiment, the first transcription factor sequence encodes apolypeptide comprising a TD, a DBD that recognizes a response elementassociated with the exogenous gene whose expression is to be modulated;and a Group H nuclear receptor LBD, and the second transcription factorsequence encodes a transcription factor comprising a nuclear receptorLBD selected from a vertebrate RXR LBD, an invertebrate RXR LBD, anultraspiracle protein LBD, and a chimeric LBD comprising two polypeptidefragments, wherein the first polypeptide fragment is from a vertebrateRXR LBD, an invertebrate RXR LBD, or an ultraspiracle protein LBD, andthe second polypeptide fragment is from a different vertebrate RXR LBD,invertebrate RXR LBD, or ultraspiracle protein LBD.

In another embodiment, the gene switch comprises a first transcriptionfactor sequence encoding a first polypeptide comprising a nuclearreceptor LBD and a DBD that recognizes a response element associatedwith the exogenous gene whose expression is to be modulated, and asecond transcription factor sequence encoding a second polypeptidecomprising a TD and a nuclear receptor LBD, wherein one of the nuclearreceptor LBDs is a Group H nuclear receptor LBD. In one embodiment, thefirst polypeptide is substantially free of a TD and the secondpolypeptide is substantially free of a DBD. For purposes of theinvention, “substantially free” means that the protein in question doesnot contain a sufficient sequence of the domain in question to provideactivation or binding activity.

In another aspect of the invention, the first transcription factorsequence encodes a protein comprising a heterodimer partner and a TD andthe second transcription factor sequence encodes a protein comprising aDBD and a LBD.

When only one nuclear receptor LBD is a Group H LBD, the other nuclearreceptor LBD may be from any other nuclear receptor that forms a dimerwith the Group H LBD. For example, when the Group H nuclear receptor LBDis an EcR LBD, the other nuclear receptor LBD “partner” may be from anEcR, a vertebrate RXR, an invertebrate RXR, an ultraspiracle protein(USP), or a chimeric nuclear receptor comprising at least two differentnuclear receptor LBD polypeptide fragments selected from a vertebrateRXR, an invertebrate RXR, and a USP (see WO 01/70816 A2, InternationalPatent Application No. PCT/US02/05235 and US 2004/0096942 A1). The“partner” nuclear receptor ligand binding domain may further comprise atruncation mutation, a deletion mutation, a substitution mutation, oranother modification.

In one embodiment, the vertebrate RXR LBD is from a human Homo sapiens,mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pigSus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio,tunicate Polyandrocarpa misakiensis, or jellyfish Tripedalia cysophoraRXR.

In one embodiment, the invertebrate RXR ligand binding domain is from alocust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodidtick Amblyomma americanum RXR homolog 1 (“AmaRXRI”), an ixodid tickAmblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celucapugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog(“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphidMyzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-LepidopteranRXR homolog.

In one embodiment, the chimeric RXR LBD comprises at least twopolypeptide fragments selected from a vertebrate species RXR polypeptidefragment, an invertebrate species RXR polypeptide fragment, and anon-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment. A chimeric RXR ligand binding domain for use inthe invention may comprise at least two different species RXRpolypeptide fragments, or when the species is the same, the two or morepolypeptide fragments may be from two or more different isoforms of thespecies RXR polypeptide fragment.

In one embodiment, the chimeric RXR ligand binding domain comprises atleast one vertebrate species RXR polypeptide fragment and oneinvertebrate species RXR polypeptide fragment.

In another embodiment, the chimeric RXR ligand binding domain comprisesat least one vertebrate species RXR polypeptide fragment and onenon-Dipteran/non-Lepidopteran invertebrate species RXR homologpolypeptide fragment.

The ligand, when combined with the LBD of the nuclear receptor(s), whichin turn are bound to the response element linked to the exogenous gene,provides external temporal regulation of expression of the exogenousgene. The binding mechanism or the order in which the various componentsof this invention bind to each other, that is, for example, ligand toLBD, DBD to response element, TD to promoter, etc., is not critical.

In a specific example, binding of the ligand to the LBD of a Group Hnuclear receptor and its nuclear receptor LBD partner enables expressionof the exogenous gene. This mechanism does not exclude the potential forligand binding to the Group H nuclear receptor (GHNR) or its partner,and the resulting formation of active homodimer complexes (e.g.,GHNR+GHNR or partner+partner). Preferably, one or more of the receptordomains is varied producing a hybrid gene switch. Typically, one or moreof the three domains, DBD, LBD, and TD, may be chosen from a sourcedifferent than the source of the other domains so that the hybrid genesand the resulting hybrid proteins are optimized in the chosen host cellor organism for transactivating activity, complementary binding of theligand, and recognition of a specific response element. In addition, theresponse element itself can be modified or substituted with responseelements for other DNA binding protein domains such as the GAL-4 proteinfrom yeast (see Sadowski et al., Nature 335:563 (1988)) or LexA proteinfrom Escherichia coli (see Brent et al., Cell 43:729 (1985)), orsynthetic response elements specific for targeted interactions withproteins designed, modified, and selected for such specific interactions(see, for example, Kim et al., Proc. Natl. Acad. Sci. USA, 94:3616(1997)) to accommodate hybrid receptors.

The functional EcR complex may also include additional protein(s) suchas immunophilins. Additional members of the nuclear receptor family ofproteins, known as transcriptional factors (such as DHR38 or betaFTZ-1),may also be ligand dependent or independent partners for EcR, USP,and/or RXR. Additionally, other cofactors may be required such asproteins generally known as coactivators (also termed adapters ormediators). These proteins do not bind sequence-specifically to DNA andare not involved in basal transcription. They may exert their effect ontranscription activation through various mechanisms, includingstimulation of DNA-binding of activators, by affecting chromatinstructure, or by mediating activator-initiation complex interactions.Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as thepromiscuous coactivator C response element B binding protein, CBP/p300(for review see Glass et al., Curr. Opin. Cell Biol. 9:222 (1997)).Also, protein cofactors generally known as corepressors (also known asrepressors, silencers, or silencing mediators) may be required toeffectively inhibit transcriptional activation in the absence of ligand.These corepressors may interact with the unliganded EcR to silence theactivity at the response element. Current evidence suggests that thebinding of ligand changes the conformation of the receptor, whichresults in release of the corepressor and recruitment of the abovedescribed coactivators, thereby abolishing their silencing activity.Examples of corepressors include N—CoR and SMRT (for review, see Horwitzet al., Mol Endocrinol. 10:1167 (1996)). These cofactors may either beendogenous within the cell or organism, or may be added exogenously astransgenes to be expressed in either a regulated or unregulated fashion.

The exogenous gene is operably linked to a promoter comprising at leastone response element that is recognized by the DBD of theligand-dependent transcription factor encoded by the gene switch. In oneembodiment, the promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore copies of the response element. Promoters comprising the desiredresponse elements may be naturally occurring promoters or artificialpromoters created using techniques that are well known in the art, e.g.,one or more response elements operably linked to a minimal promoter.

A gene encoding a protein can also be codon-optimized. In oneembodiment, a coding region of a protein is codon-optimized forexpression in human. As appreciated by one of ordinary skill in the art,various nucleic acid coding regions will encode the same polypeptide dueto the redundancy of the genetic code. Deviations in the nucleotidesequence that comprise the codons encoding the amino acids of anypolypeptide chain allow for variations in the sequence coding for thegene. Since each codon consists of three nucleotides, and thenucleotides comprising DNA are restricted to four specific bases, thereare 64 possible combinations of nucleotides, 61 of which encode aminoacids (the remaining three codons encode signals ending translation).The “genetic code” which shows which codons encode which amino acids isreproduced herein as Table 4. As a result, many amino acids aredesignated by more than one codon. For example, the amino acids alanineand proline are coded for by four triplets, serine and arginine by six,whereas tryptophan and methionine are coded by just one triplet. Thisdegeneracy allows for DNA base composition to vary over a wide rangewithout altering the amino acid sequence of the polypeptides encoded bythe DNA.

TABLE 4 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TATTyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L)TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W)C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro(P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg(R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACTThr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGCSer (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACGThr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D)GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V)GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E)GGG Gly (G)

It is to be appreciated that any polynucleotide that encodes apolypeptide in accordance with the invention falls within the scope ofthis invention, regardless of the codons used.

Many organisms display a bias for use of particular codons to code forinsertion of a particular amino acid in a growing polypeptide chain.Codon preference or codon bias, differences in codon usage betweenorganisms, is afforded by degeneracy of the genetic code, and is welldocumented among many organisms. Codon bias often correlates with theefficiency of translation of messenger RNA (mRNA), which is in turnbelieved to be dependent on, inter alia, the properties of the codonsbeing translated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization.

The polynucleotides are prepared by incorporating codons preferred foruse in the genes of a given species into the DNA sequence.

Given the large number of gene sequences available for a wide variety ofanimal, plant and microbial species, it is possible to calculate therelative frequencies of codon usage. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database” available athttp://www.kazusa.or.jp/codon/ (visited May 30, 2006), and these tablescan be adapted in a number of ways. See Nakamura, Y., et al., “Codonusage tabulated from the international DNA sequence databases: statusfor the year 2000” Nucl. Acids Res. 28:292 (2000). Codon usage tablesfor humans calculated from GenBank Release 151.0, are reproduced belowas Table 5 (from http://www.kazusa.or.jp/codon/supra). These tables usemRNA nomenclature, and so instead of thymine (T) which is found in DNA,the tables use uracil (U) which is found in RNA. The tables have beenadapted so that frequencies are calculated for each amino acid, ratherthan for all 64 codons.

TABLE 5 Codon Usage Table for Human Genes (Homo sapiens) Amino AcidCodon Frequency of Usage Phe UUU 0.4525 UUC 0.5475 Leu UUA 0.0728 UUG0.1266 CUU 0.1287 CUC 0.1956 CUA 0.0700 CUG 0.4062 Ile AUU 0.3554 AUC0.4850 AUA 0.1596 Met AUG 1.0000 Val GUU 0.1773 GUC 0.2380 GUA 0.1137GUG 0.4710 Ser UCU 0.1840 UCC 0.2191 UCA 0.1472 UCG 0.0565 AGU 0.1499AGC 0.2433 Pro CCU 0.2834 CCC 0.3281 CCA 0.2736 CCG 0.1149 Thr ACU0.2419 ACC 0.3624 ACA 0.2787 ACG 0.1171 Ala GCU 0.2637 GCC 0.4037 GCA0.2255 GCG 0.1071 Tyr UAU 0.4347 UAC 0.5653 His CAU 0.4113 CAC 0.5887Gln CAA 0.2541 CAG 0.7459 Asn AAU 0.4614 AAC 0.5386 Lys AAA 0.4212 AAG0.5788 Asp GAU 0.4613 GAC 0.5387 Glu GAA 0.4161 GAG 0.5839 Cys UGU0.4468 UGC 0.5532 Trp UGG 1.0000 Arg CGU 0.0830 CGC 0.1927 CGA 0.1120CGG 0.2092 AGA 0.2021 AGG 0.2011 Gly GGU 0.1632 GGC 0.3438 GGA 0.2459GGG 0.2471

By utilizing these or similar tables, one of ordinary skill in the artcan apply the frequencies to any given polypeptide sequence, and producea nucleic acid fragment of a codon-optimized coding region which encodesthe polypeptide, but which uses codons optimal for a given species.

A number of options are available for synthesizing codon-optimizedcoding regions designed by any of the methods described above, usingstandard and routine molecular biological manipulations well known tothose of ordinary skill in the art.

In one embodiment, the coding region encoding the protein in the vectorof the invention is codon-optimized. In another embodiment, the codingregion is codon-optimized for expression in human. In a particularembodiment, the sequence is a codon-optimized nucleic acid sequence.

To introduce the polynucleotides into the cells in vivo or ex vivo, avector can be used. The vector may be, for example, a plasmid vector ora single- or double-stranded RNA or DNA viral vector. Such vectors maybe introduced into cells of a subject in need thereof, e.g., mammal, bywell-known techniques for introducing DNA and RNA into cells. Viralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells. As used herein, the term “host cell” or “host”is used to mean a cell of the invention that is harboring one or morepolynucleotides of the invention.

Thus, at a minimum, the vectors must include the polynucleotides of theinvention. Other components of the vector may include, but are notlimited to, selectable markers, chromatin modification domains,additional promoters driving expression of other polypeptides that mayalso be present on the vector (e.g., a lethal polypeptide), genomicintegration sites, recombination sites, and molecular insertion pivots.The vectors may comprise any number of these additional elements, eitherwithin or not within the polynucleotides, such that the vector can betailored to the specific goals of the therapeutic methods desired.

In one embodiment of the invention, the vectors that are introduced intothe cells further comprise a “selectable marker gene” which, whenexpressed, indicates that the gene switch construct of the invention hasbeen integrated into the genome of the host cell. In this manner, theselector gene can be a positive marker for the genome integration. Whilenot critical to the methods of the invention, the presence of aselectable marker gene allows the practitioner to select for apopulation of live cells where the vector construct has been integratedinto the genome of the cells. Thus, certain embodiments of the inventioncomprise selecting cells where the vector has successfully beenintegrated. As used herein, the term “select” or variations thereof,when used in conjunction with cells, is intended to mean standard,well-known methods for choosing cells with a specific genetic make-up orphenotype. Typical methods include, but are not limited to, culturingcells in the presence of antibiotics, such as G418, neomycin andampicillin. Other examples of selectable marker genes include, but arenot limited to, genes that confer resistance to dihydrofolate reductase,hygromycin, or mycophenolic acid. Other methods of selection include,but are not limited to, a selectable marker gene that allows for the useof thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase oradenine phosphoribosyltransferase as selection agents. Cells comprisinga vector construct comprising an antibiotic resistance gene or geneswould then be capable of tolerating the antibiotic in culture. Likewise,cells not comprising a vector construct comprising an antibioticresistance gene or genes would not be capable of tolerating theantibiotic in culture.

As used herein, a “chromatin modification domain” (CMD) refers tonucleotide sequences that interact with a variety of proteins associatedwith maintaining and/or altering chromatin structure, such as, but notlimited to, DNA insulators. See Ciavatta et al., Proc. Nat'l Acad. Sci.U.S.A., 103:9958 (2006). Examples of CMDs include, but are not limitedto, the chicken β-globulin insulator and the chicken hypersensitive site4 (cHS4). The use of different CMD sequences between one or more geneprograms (i.e., a promoter, coding sequence, and 3′ regulatory region),for example, can facilitate the use of the differential CMD DNAsequences as “mini homology arms” in combination with variousmicroorganism or in vitro recombineering technologies to “swap” geneprograms between existing multigenic and monogenic shuttle vectors.Other examples of chromatin modification domains are known in the art orcan be readily identified.

Polynucleotide and nucleic acid coding regions in the vector of theinvention can be associated with additional coding regions which encodesecretory or signal peptides, which direct the secretion of a protein.According to the signal hypothesis, proteins secreted by mammalian cellshave a signal peptide or secretory leader sequence which is cleaved fromthe mature protein once export of the growing protein chain across therough endoplasmic reticulum has been initiated. Polypeptides secreted byvertebrate cells generally have a signal peptide fused to the N-terminusof the polypeptide, which is cleaved from the complete or “full length”polypeptide to produce a secreted or “mature” form of the polypeptide.

In one embodiment, a vector of the invention comprises a polynucleotideencoding a gene switch, wherein said polynucleotide comprises (1) atleast one transcription factor sequence which is operably linked to apromoter, wherein said at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and (2) apolynucleotide encoding one or more proteins operably linked to apromoter which is activated by said ligand-dependent transcriptionfactor, wherein said polynucleotide encoding one or more proteinsfurther comprises a nucleic acid sequence encoding a signal peptide. Inanother embodiment, the signal peptide increases secretion of theprotein encoded by the vector, compared to a vector comprising theprotein's native signal peptide gene. In particular, the signal peptideused in the invention can be codon-optimized.

The vector of the invention can comprise various regulatory regions, forexample, 5′ untranslated region (5′UTR), 3′ UTR, or both. The presentinvention is also directed to using various regulatory regions to induceimproved secretion, protein translation, post-translation, mRNAtranscription, or post-transcription process. As used herein, the “5′untranslated region” or “5′UTR” of a gene is to be understood as thatpart of a gene which is transcribed into a primary RNA transcript(pre-mRNA) and which part is located upstream of the coding sequence.The primary transcript is the initial RNA product, containing intronsand exons, produced by transcription of DNA. Many primary transcriptsmust undergo RNA processing to form the physiologically active RNAspecies. The processing into a mature mRNA may comprise trimming of theends, removal of introns, capping and/or cutting out of individual rRNAmolecules from their precursor RNAs. The 5′UTR of an mRNA is thus thatpart of the mRNA which is not translated into protein and which islocated upstream of the coding sequence. In a genomic sequence, the5′UTR is typically defined as the region between the transcriptioninitiation site and the start codon. The 5′ untranslated regions(5′UTRs) of vertebrate mRNAs may be a few tens of bases to severalhundred bases in length (Crowe et al., 2006 BMC Genomics 7:16). The5′UTR used herein may occur naturally or be modified to contain one ormore nucleic acid sequences not contiguous in nature (chimericsequences), and/or may encompass substitutions, insertions, anddeletions and combinations thereof. In one embodiment, the 5′UTRsequence is derived from the wild-type TNF-alpha sequence or 5U2sequence. In another embodiment, the 5′UTR sequence is 5′UTR of 5U2. Insome embodiments, the 5′UTR induces improved protein expression, e.g,mRNA transcription, pre-transcription, or post-transcription.

The 3′ untranslated region (UTR) used in the invention refer to DNAsequences located downstream (3′) of a coding sequence and may comprisepolyadenylation [poly(A)] recognition sequences and other sequencesencoding regulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. Any suitable polyadenylation sequence can be used,including a synthetic optimized sequence, as well as the polyadenylationsequence of BGH (Bovine Growth Hormone), polyoma virus, TK (ThymidineKinase), EBV (Epstein Barr Virus), and the papillomaviruses, includinghuman papillomaviruses and BPV (Bovine Papilloma Virus). In a particularembodiment, a 3′ regulatory region is the SV40e (human Sarcoma Virus-40)polyadenylation sequence. In another particular embodiment, a 3′regulatory region is the polyadenylation sequence of human growthhormone.

In certain embodiments, the signal peptide and/or the regulatory regionalone or in combination can improve the protein secretion,transcription, or translation at least two fold, three fold, four fold,five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 50fold, 100 fold, 200 fold, 300 fold, 400 fold, or 500 fold compared to acontrol, which does not contain the signal peptide and/or the regulatoryregion. The secretion level of a protein, e.g., TNF-alpha, can benormalized to the protein expression encoded by a vector having awild-type gene. In another specific embodiment of the present invention,the signal peptide and/or the regulatory region alone or in combinationincrease productivity of the protein about 5% to about 10%, about 11% toabout 20%, about 21% to about 30%, about 31% to about 40%, about 41% toabout 50%, about 51% to about 60%, about 61% to about 70%, about 71% toabout 80%, about 81% to about 90%, about 91% to about 100%, about 101%to about 149%, about 150% to about 199%, about 200% to about 299%, about300% to about 499%, or about 500% to about 1000%. In a specificembodiment, the present invention comprises a vector conditionallyexpressing a protein wherein said vector comprises 5′ UTR of 5U2, acodon-optimized nucleic acid sequence encoding IL-2 signal peptide, acodon-optimized coding region encoding a protein and a polyadenylationsignal of SV40e or human growth hormone.

Particular vectors for use with the invention are expression vectorsthat code for proteins or polynucleotides. Generally, such vectorscomprise cis-acting control regions effective for expression in a hostoperatively linked to the polynucleotide to be expressed. Appropriatetrans-acting factors are supplied by the host, supplied by acomplementing vector or supplied by the vector itself upon introductioninto the host.

A great variety of expression vectors can be used to express proteins orpolynucleotides. Such vectors include chromosomal, episomal andvirus-derived vectors, e.g., vectors derived from bacterial plasmids,from bacteriophage, from yeast episomes, from yeast chromosomalelements, from viruses such as adeno-associated viruses, lentiviruses,baculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids. All may be used for expression in accordance with this aspectof the invention. Generally, any vector suitable to maintain, propagateor express polynucleotides or proteins in a host may be used forexpression in this regard.

Suitable viral vectors used in the invention include, but not limitedto, adenovirus-based vectors, retroviral vectors, herpes simplex virus(HSV)-based vectors, parvovirus-based vectors, e.g., adeno-associatedvirus (AAV)-based vectors, and AAV-adenoviral chimeric vectors. Theseviral vectors can be prepared using standard recombinant DNA techniquesdescribed in, for example, Sambrook et al., Molecular Cloning, aLaboratory Manual, 2d edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N.Y. (1994).

In one embodiment, a viral vector of the invention is an adenoviralvector. Adenovirus (Ad) is a 36 kb double-stranded DNA virus thatefficiently transfers DNA in vivo to a variety of different target celltypes. The adenoviral vector can be produced in high titers and canefficiently transfer DNA to replicating and non-replicating cells. Theadenoviral vector genome can be generated using any species, strain,subtype, mixture of species, strains, or subtypes, or chimericadenovirus as the source of vector DNA. Adenoviral stocks that can beemployed as a source of adenovirus can be amplified from the adenoviralserotypes 1 through 51, which are currently available from the AmericanType Culture Collection (ATCC, Manassas, Va.), or from any otherserotype of adenovirus available from any other source. For instance, anadenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31),subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroupC (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9,10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E(serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviralserotype. Given that the human adenovirus serotype 5 (Ad5) genome hasbeen completely sequenced, the adenoviral vector of the invention isdescribed herein with respect to the Ad5 serotype. The adenoviral vectorcan be any adenoviral vector capable of growth in a cell, which is insome significant part (although not necessarily substantially) derivedfrom or based upon the genome of an adenovirus. The adenoviral vectorcan be based on the genome of any suitable wild-type adenovirus. Incertain embodiments, the adenoviral vector is derived from the genome ofa wild-type adenovirus of group C, especially of serotype 2 or 5.Adenoviral vectors are well known in the art and are described in, forexample, U.S. Pat. Nos. 5,559,099, 5,712,136, 5,731,190, 5,837,511,5,846,782, 5,851,806, 5,962,311, 5,965,541, 5,981,225, 5,994,106,6,020,191, and 6,113,913, International Patent Applications WO 95/34671,WO 97/21826, and WO 00/00628, and Thomas Shenk, “Adenoviridae and theirReplication,” and M. S. Horwitz, “Adenoviruses,” Chapters 67 and 68,respectively, in Virology, B. N. Fields et al., eds., 3d ed., RavenPress, Ltd., New York (1996).

In other embodiments, the adenoviral vector is replication-deficient.The term “replication-deficient” used herein means that the adenoviralvector comprises a genome that lacks at least one replication-essentialgene function. A deficiency in a gene, gene function, or gene or genomicregion, as used herein, is defined as a deletion of sufficient geneticmaterial of the viral genome to impair or obliterate the function of thegene whose nucleic acid sequence was deleted in whole or in part.Replication-essential gene functions are those gene functions that arerequired for replication (i.e., propagation) of a replication-deficientadenoviral vector. Replication-essential gene functions are encoded by,for example, the adenoviral early regions (e.g., the E1, E2, and E4regions), late regions (e.g., the L1-L5 regions), genes involved inviral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g.,VA-RNA I and/or VA-RNA II). In still other embodiments, thereplication-deficient adenoviral vector comprises an adenoviral genomedeficient in at least one replication-essential gene function of one ormore regions of an adenoviral genome (e.g., two or more regions of anadenoviral genome so as to result in a multiply replication-deficientadenoviral vector). The one or more regions of the adenoviral genome areselected from the group consisting of the E1, E2, and E4 regions. Thereplication-deficient adenoviral vector can comprise a deficiency in atleast one replication-essential gene function of the E1 region (denotedan E1-deficient adenoviral vector), particularly a deficiency in areplication-essential gene function of each of the adenoviral E1A regionand the adenoviral E1B region. In addition to such a deficiency in theE1 region, the recombinant adenovirus also can have a mutation in themajor late promoter (MLP), as discussed in International PatentApplication WO 00/00628. In a particular embodiment, the vector isdeficient in at least one replication-essential gene function of the E1region and at least part of the nonessential E3 region (e.g., an Xba Ideletion of the E3 region) (denoted an E1/E3-deficient adenoviralvector).

In certain embodiments, the adenoviral vector is “multiply deficient,”meaning that the adenoviral vector is deficient in one or more genefunctions required for viral replication in each of two or more regionsof the adenoviral genome. For example, the aforementioned E1-deficientor E1/E3-deficient adenoviral vector can be further deficient in atleast one replication-essential gene function of the E4 region (denotedan E1/E4-deficient adenoviral vector). An adenoviral vector deleted ofthe entire E4 region can elicit a lower host immune response.

Alternatively, the adenoviral vector lacks replication-essential genefunctions in all or part of the E1 region and all or part of the E2region (denoted an E1/E2-deficient adenoviral vector). Adenoviralvectors lacking replication-essential gene functions in all or part ofthe E1 region, all or part of the E2 region, and all or part of the E3region also are contemplated herein. If the adenoviral vector of theinvention is deficient in a replication-essential gene function of theE2A region, the vector does not comprise a complete deletion of the E2Aregion, which is less than about 230 base pairs in length. Generally,the E2A region of the adenovirus codes for a DBP (DNA binding protein),a polypeptide required for DNA replication. DBP is composed of 473 to529 amino acids depending on the viral serotype. It is believed that DBPis an asymmetric protein that exists as a prolate ellipsoid consistingof a globular Ct with an extended Nt domain. Studies indicate that theCt domain is responsible for DBP's ability to bind to nucleic acids,bind to zinc, and function in DNA synthesis at the level of DNA chainelongation. However, the Nt domain is believed to function in late geneexpression at both transcriptional and post-transcriptional levels, isresponsible for efficient nuclear localization of the protein, and alsomay be involved in enhancement of its own expression. Deletions in theNt domain between amino acids 2 to 38 have indicated that this region isimportant for DBP function (Brough et al., Virology, 196, 269-281(1993)). While deletions in the E2A region coding for the Ct region ofthe DBP have no effect on viral replication, deletions in the E2A regionwhich code for amino acids 2 to 38 of the Nt domain of the DBP impairviral replication. In one embodiment, the multiply replication-deficientadenoviral vector contains this portion of the E2A region of theadenoviral genome. In particular, for example, the desired portion ofthe E2A region to be retained is that portion of the E2A region of theadenoviral genome which is defined by the 5′ end of the E2A region,specifically positions Ad5(23816) to Ad5(24032) of the E2A region of theadenoviral genome of serotype Ad5.

The adenoviral vector can be deficient in replication-essential genefunctions of only the early regions of the adenoviral genome, only thelate regions of the adenoviral genome, and both the early and lateregions of the adenoviral genome. The adenoviral vector also can haveessentially the entire adenoviral genome removed, in which case at leasteither the viral inverted terminal repeats (ITRs) and one or morepromoters or the viral ITRs and a packaging signal are left intact(i.e., an adenoviral amplicon). The larger the region of the adenoviralgenome that is removed, the larger the piece of exogenous nucleic acidsequence that can be inserted into the genome. For example, given thatthe adenoviral genome is 36 kb, by leaving the viral ITRs and one ormore promoters intact, the exogenous insert capacity of the adenovirusis approximately 35 kb. Alternatively, a multiply deficient adenoviralvector that contains only an ITR and a packaging signal effectivelyallows insertion of an exogenous nucleic acid sequence of approximately37-38 kb. Of course, the inclusion of a spacer element in any or all ofthe deficient adenoviral regions will decrease the capacity of theadenoviral vector for large inserts. Suitable replication-deficientadenoviral vectors, including multiply deficient adenoviral vectors, aredisclosed in U.S. Pat. Nos. 5,851,806 and 5,994,106 and InternationalPatent Applications WO 95/34671 and WO 97/21826. In one embodiment, thevector for use in the present inventive method is that described inInternational Patent Application PCT/US01/20536.

It should be appreciated that the deletion of different regions of theadenoviral vector can alter the immune response of the mammal. Inparticular, the deletion of different regions can reduce theinflammatory response generated by the adenoviral vector. Furthermore,the adenoviral vector's coat protein can be modified so as to decreasethe adenoviral vector's ability or inability to be recognized by aneutralizing antibody directed against the wild-type coat protein, asdescribed in International Patent Application WO 98/40509.

The adenoviral vector, when multiply replication-deficient, especiallyin replication-essential gene functions of the E1 and E4 regions, caninclude a spacer element to provide viral growth in a complementing cellline similar to that achieved by singly replication deficient adenoviralvectors, particularly an adenoviral vector comprising a deficiency inthe E1 region. The spacer element can contain any sequence or sequenceswhich are of the desired length. The spacer element sequence can becoding or non-coding and native or non-native with respect to theadenoviral genome, but does not restore the replication-essentialfunction to the deficient region. In the absence of a spacer, productionof fiber protein and/or viral growth of the multiplyreplication-deficient adenoviral vector is reduced by comparison to thatof a singly replication-deficient adenoviral vector. However, inclusionof the spacer in at least one of the deficient adenoviral regions,preferably the E4 region, can counteract this decrease in fiber proteinproduction and viral growth. The use of a spacer in an adenoviral vectoris described in U.S. Pat. No. 5,851,806.

Construction of adenoviral vectors is well understood in the art.Adenoviral vectors can be constructed and/or purified using the methodsset forth, for example, in U.S. Pat. No. 5,965,358 and InternationalPatent Applications WO 98/56937, WO 99/15686, and WO 99/54441. Theproduction of adenoviral gene transfer vectors is well known in the art,and involves using standard molecular biological techniques such asthose described in, for example, Sambrook et al., supra, Watson et al.,supra, Ausubel et al., supra, and in several of the other referencesmentioned herein.

Replication-deficient adenoviral vectors are typically produced incomplementing cell lines that provide gene functions not present in thereplication-deficient adenoviral vectors, but required for viralpropagation, at appropriate levels in order to generate high titers ofviral vector stock. In one embodiment, a cell line complements for atleast one and/or all replication-essential gene functions not present ina replication-deficient adenovirus. The complementing cell line cancomplement for a deficiency in at least one replication-essential genefunction encoded by the early regions, late regions, viral packagingregions, virus-associated RNA regions, or combinations thereof,including all adenoviral functions (e.g., to enable propagation ofadenoviral amplicons, which comprise minimal adenoviral sequences, suchas only inverted terminal repeats (ITRs) and the packaging signal oronly ITRs and an adenoviral promoter). In another embodiment, thecomplementing cell line complements for a deficiency in at least onereplication-essential gene function (e.g., two or morereplication-essential gene functions) of the E1 region of the adenoviralgenome, particularly a deficiency in a replication-essential genefunction of each of the E1A and E1B regions. In addition, thecomplementing cell line can complement for a deficiency in at least onereplication-essential gene function of the E2 (particularly as concernsthe adenoviral DNA polymerase and terminal protein) and/or E4 regions ofthe adenoviral genome. Desirably, a cell that complements for adeficiency in the E4 region comprises the E4-ORF6 gene sequence andproduces the E4-ORF6 protein. Such a cell desirably comprises at leastORF6 and no other ORF of the E4 region of the adenoviral genome. Thecell line preferably is further characterized in that it contains thecomplementing genes in a non-overlapping fashion with the adenoviralvector, which minimizes, and practically eliminates, the possibility ofthe vector genome recombining with the cellular DNA. Accordingly, thepresence of replication competent adenoviruses (RCA) is minimized if notavoided in the vector stock, which, therefore, is suitable for certaintherapeutic purposes, especially gene therapy purposes. The lack of RCAin the vector stock avoids the replication of the adenoviral vector innon-complementing cells. The construction of complementing cell linesinvolves standard molecular biology and cell culture techniques, such asthose described by Sambrook et al., supra, and Ausubel et al., supra.Complementing cell lines for producing the gene transfer vector (e.g.,adenoviral vector) include, but are not limited to, 293 cells (describedin, e.g., Graham et al., J. Gen. Virol., 36, 59-72 (1977)), PER.C6 cells(described in, e.g., International Patent Application WO 97/00326, andU.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (describedin, e.g., International Patent Application WO 95/34671 and Brough etal., J. Virol., 71, 9206-9213 (1997)). The insertion of a nucleic acidsequence into the adenoviral genome (e.g., the E1 region of theadenoviral genome) can be facilitated by known methods, for example, bythe introduction of a unique restriction site at a given position of theadenoviral genome.

Retrovirus is an RNA virus capable of infecting a wide variety of hostcells. Upon infection, the retroviral genome integrates into the genomeof its host cell and is replicated along with host cell DNA, therebyconstantly producing viral RNA and any nucleic acid sequenceincorporated into the retroviral genome. As such, long-term expressionof a therapeutic factor(s) is achievable when using retrovirus.Retroviruses contemplated for use in gene therapy are relativelynon-pathogenic, although pathogenic retroviruses exist. When employingpathogenic retroviruses, e.g., human immunodeficiency virus (HIV) orhuman T-cell lymphotrophic viruses (HTLV), care must be taken inaltering the viral genome to eliminate toxicity to the host. Aretroviral vector additionally can be manipulated to render the virusreplication-deficient. As such, retroviral vectors are consideredparticularly useful for stable gene transfer in vivo. Lentiviralvectors, such as HIV-based vectors, are exemplary of retroviral vectorsused for gene delivery. Unlike other retroviruses, HIV-based vectors areknown to incorporate their passenger genes into non-dividing cells and,therefore, can be of use in treating persistent forms of disease.

An HSV-based viral vector is suitable for use as a gene transfer vectorto introduce a nucleic acid into numerous cell types. The mature HSVvirion consists of an enveloped icosahedral capsid with a viral genomeconsisting of a linear double-stranded DNA molecule that is 152 kb. Mostreplication-deficient HSV vectors contain a deletion to remove one ormore intermediate-early genes to prevent replication. Advantages of theHSV vector are its ability to enter a latent stage that can result inlong-term DNA expression and its large viral DNA genome that canaccommodate exogenous DNA inserts of up to 25 kb. Of course, the abilityof HSV to promote long-term production of exogenous protein ispotentially disadvantageous in terms of short-term treatment regimens.However, one of ordinary skill in the art has the requisiteunderstanding to determine the appropriate vector for a particularsituation. HSV-based vectors are described in, for example, U.S. Pat.Nos. 5,837,532, 5,846,782, 5,849,572, and 5,804,413, and InternationalPatent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO99/06583.

AAV vectors are viral vectors of particular interest for use in genetherapy protocols. AAV is a DNA virus, which is not known to cause humandisease. The AAV genome is comprised of two genes, rep and cap, flankedby inverted terminal repeats (ITRs), which contain recognition signalsfor DNA replication and packaging of the virus. AAV requiresco-infection with a helper virus (i.e., an adenovirus or a herpessimplex virus), or expression of helper genes, for efficientreplication. AAV can be propagated in a wide array of host cellsincluding human, simian, and rodent cells, depending on the helper virusemployed. An AAV vector used for administration of a nucleic acidsequence typically has approximately 96% of the parental genome deleted,such that only the ITRs remain. This eliminates immunologic or toxicside effects due to expression of viral genes. If desired, the AAV repprotein can be co-administered with the AAV vector to enable integrationof the AAV vector into the host cell genome. Host cells comprising anintegrated AAV genome show no change in cell growth or morphology (see,e.g., U.S. Pat. No. 4,797,368). As such, prolonged expression oftherapeutic factors from AAV vectors can be useful in treatingpersistent and chronic diseases.

The polynucleotide sequence in the expression vector is operativelylinked to appropriate expression control sequence(s) including, forinstance, a promoter to direct mRNA transcription. Representatives ofadditional promoters include, but are not limited to, constitutivepromoters and tissue specific or inducible promoters. Examples ofconstitutive eukaryotic promoters include, but are not limited to, thepromoter of the mouse metallothionein I gene (Hamer et al., J. Mol.Appl. Gen. 1:273 (1982)); the TK promoter of Herpes virus (McKnight,Cell 31:355 (1982)); the SV40 early promoter (Benoist et al., Nature290:304 (1981)); and the vaccinia virus promoter. Additional examples ofthe promoters that could be used to drive expression of a protein orpolynucleotide include, but are not limited to, tissue-specificpromoters and other endogenous promoters for specific proteins, such asthe albumin promoter (hepatocytes), a proinsulin promoter (pancreaticbeta cells) and the like. In general, expression constructs will containsites for transcription, initiation and termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the mature transcripts expressed by the constructs mayinclude a translation initiating AUG at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

In addition, the constructs may contain control regions that regulate,as well as engender expression. Generally, such regions will operate bycontrolling transcription, such as repressor binding sites andenhancers, among others.

Examples of eukaryotic vectors include, but are not limited to, pW-LNEO,pSV2CAT, pOG44, pXTI and pSG available from Stratagene; pSVK3, pBPV,pMSG and pSVL available from Amersham Pharmacia Biotech; andpCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP availablefrom Clontech. Many other vectors are well-known and commerciallyavailable.

Particularly useful vectors, which comprise molecular insertion pivotsfor rapid insertion and removal of elements of gene programs, aredescribed in United States Published Patent Application No.2004/0185556, U.S. patent application Ser. No. 11/233,246 andInternational Published Application Nos. WO 2005/040336 and WO2005/116231. An example of such vectors is the UltraVector™ ProductionSystem (Intrexon Corp., Blacksburg, Va.), as described in WO2007/038276. As used herein, a “gene program” is a combination ofgenetic elements comprising a promoter (P), an expression sequence (E)and a 3′ regulatory sequence (3), such that “PE3” is a gene program. Theelements within the gene program can be easily swapped between molecularpivots that flank each of the elements of the gene program. A molecularpivot, as used herein, is defined as a polynucleotide comprising atleast two non-variable rare or uncommon restriction sites arranged in alinear fashion. In one embodiment, the molecular pivot comprises atleast three non-variable rare or uncommon restriction sites arranged ina linear fashion. Typically any one molecular pivot would not include arare or uncommon restriction site of any other molecular pivot withinthe same gene program. Cognate sequences of greater than 6 nucleotidesupon which a given restriction enzyme acts are referred to as “rare”restriction sites. There are, however, restriction sites of 6 bp thatoccur more infrequently than would be statistically predicted, and thesesites and the endonucleases that cleave them are referred to as“uncommon” restriction sites. Examples of either rare or uncommonrestriction enzymes include, but are not limited to, AsiS I, Pac I, SbfI, Fse I, Asc I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, SfoI, Sgr AI, AflIII, Pvu I, Ngo MIV, Ase I, Flp I, Pme I, Sda I, SgfI, SrfI, Nru I, Acl I, Cla I, Csp45 I, Age I, Bst1107 I, BstB I, Hpa I, AatII, EcoR V, Nhe I, Spe I, Avi II, Avr II, Mfe I, Afe I, Fsp I, Kpn I,Sca I, BspE I, Nde I, Bfr I, Xho I, Pml I, ApaL I, Kas I, Xma I, BsrB I,Nsi I, Sac II, Sac I, Blp I, PspoM I, Pci I, Stu I, Sph I, BamH I, Bsu36I, Xba I, BbvC I, Bgl II, Nco I, Hind III, EcoR I, BsrG I and Sse8781 I.

The vector may also comprise restriction sites for a second class ofrestriction enzymes called homing endonuclease (HE) enzymes. HE enzymeshave large, asymmetric restriction sites (12-40 base pairs), and theirrestriction sites are infrequent in nature. For example, the HE known asI-SceI has an 18 bp restriction site (5′TAGGGATAACAGGGTAAT3′), predictedto occur only once in every 7×10¹⁰ base pairs of random sequence. Thisrate of occurrence is equivalent to only one site in a genome that is 20times the size of a mammalian genome. The rare nature of HE sitesgreatly increases the likelihood that a genetic engineer can cut a geneprogram without disrupting the integrity of the gene program if HE sitesare included in appropriate locations in a cloning vector plasmid.

Selection of appropriate vectors and promoters for expression in a hostcell is a well-known procedure, and the requisite techniques for vectorconstruction and introduction into the host, as well as its expressionin the host are routine skills in the art.

The introduction of the polynucleotides into the cells can be atransient transfection, stable transfection, or can be a locus-specificinsertion of the vector. Transient and stable transfection of thevectors into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods in Molecular Biology(1986); Keown et al., 1990, Methods Enzymol. 185: 527-37; Sambrook etal., 2001, Molecular Cloning, A Laboratory Manual, Third Edition, ColdSpring Harbor Laboratory Press, N.Y. These stable transfection methodsresult in random insertion of the vector into the genome of the cell.Further, the copy number and orientation of the vectors are also,generally speaking, random.

In one embodiment of the invention, the vector is inserted into abio-neutral site in the genome. A bio-neutral site is a site in thegenome where insertion of the polynucleotides interferes very little, ifany, with the normal function of the cell. Bio-neutral sites may beanalyzed using available bioinformatics. Many bio-neutral sites areknown in the art, e.g., the ROSA-equivalent locus. Other bio-neutralsites may be identified using routine techniques well known in the art.Characterization of the genomic insertion site(s) is performed usingmethods known in the art. To control the location, copy number and/ororientation of the polynucleotides when introducing the vector into thecells, methods of locus-specific insertion may be used. Methods oflocus-specific insertion are well-known in the art and include, but arenot limited to, homologous recombination and recombinase-mediated genomeinsertion. Of course, if locus-specific insertion methods are to be usedin the methods of the invention, the vectors may comprise elements thataid in this locus-specific insertion, such as, but not limited to,homologous recombination. For example, the vectors may comprise one,two, three, four or more genomic integration sites (GISs). As usedherein, a “genomic integration site” is defined as a portion of thevector sequence which nucleotide sequence is identical or nearlyidentical to portions of the genome within the cells that allows forinsertion of the vector in the genome. In particular, the vector maycomprise two genomic insertion sites that flank at least thepolynucleotides. Of course, the GISs may flank additional elements, oreven all elements present on the vector.

In another embodiment, locus-specific insertion may be carried out byrecombinase-site specific gene insertion. Briefly, bacterial recombinaseenzymes, such as, but not limited to, PhiC31 integrase can act on“pseudo” recombination sites within the human genome. These pseudorecombination sites can be targets for locus-specific insertion usingthe recombinases. Recombinase-site specific gene insertion is describedin Thyagarajan et al., Mol. Cell Biol. 21:3926 (2001). Other examples ofrecombinases and their respective sites that may be used forrecombinase-site specific gene insertion include, but are not limitedto, serine recombinases such as R4 and TP901-1 and recombinasesdescribed in WO 2006/083253.

In a further embodiment, the vector may comprise a chemo-resistancegene, e.g., the multidrug resistance gene mdrl, dihydrofolate reductase,or O⁶-alkylguanine-DNA alkyltransferase. The chemo-resistance gene maybe under the control of a constitutive (e.g., CMV) or inducible (e.g.,RheoSwitch®) promoter. In this embodiment, if it is desired to treat adisease in a subject while maintaining the modified cells within thesubject, a clinician may apply a chemotherapeutic agent to destroydiseased cells while the modified cells would be protected from theagent due to expression of a suitable chemo-resistance gene and maycontinue to be used for treatment, amelioration, or prevention of adisease or disorder. By placing the chemo-resistance gene under aninducible promoter, the unnecessary expression of the chemo-resistancegene can be avoided, yet it will still be available in case continuedtreatment is needed. If the modified cells themselves become diseased,they could still be destroyed by inducing expression of a lethalpolypeptide as described below.

The methods of the invention are carried out by introducing thepolynucleotides encoding the gene switch and the exogenous gene intocells of a subject. Any method known for introducing a polynucleotideinto a cell known in the art, such as those described above, can beused.

When the polynucleotides are to be introduced into cells ex vivo, thecells may be obtained from a subject by any technique known in the art,including, but not limited to, biopsies, scrapings, and surgical tissueremoval. The isolated cells may be cultured for a sufficient amount oftime to allow the polynucleotides to be introduced into the cells, e.g.,2, 4, 6, 8, 10, 12, 18, 24, 36, 48, hours or more. Methods for culturingprimary cells for short periods of time are well known in the art. Forexample, cells may be cultured in plates (e.g., in microwell plates)either attached or in suspension.

For ex vivo therapeutic methods, cells are isolated from a subject andcultured under conditions suitable for introducing the polynucleotidesinto the cells. Once the polynucleotides have been introduced into thecells, the cells are incubated for a sufficient period of time to allowthe ligand-dependent transcription factor to be expressed, e.g., 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours or more. At some pointafter the introduction of the polynucleotides into the cells (eitherbefore or after significant levels of the ligand-dependent transcriptionfactor is expressed), the cells are introduced back into the subject.Reintroduction may be carried out by any method known in the art, e.g.,intravenous infusion or direct injection into a tissue or cavity. In oneembodiment, the presence of the polynucleotides in the cells isdetermined prior to introducing the cells back into the subject. Inanother embodiment, cells containing the polynucleotides are selected(e.g., based on the presence of a selectable marker in thepolynucleotides) and only those cells containing the polynucleotides arereintroduced into the subject. After the cells are reintroduced to thesubject, ligand is administered to the subject to induce expression ofthe therapeutic polypeptide or therapeutic polynucleotide. In analternative embodiment, the ligand may be added to the cells even beforethe cells are reintroduced to the subject such that the therapeuticpolypeptide or therapeutic polynucleotide is expressed prior toreintroduction of the cells. The ligand may be administered by anysuitable method, either systemically (e.g., orally, intravenously) orlocally (e.g., intraperitoneally, intrathecally, intraventricularly,direct injection into the tissue or organ where the cells arereintroduced). The optimal timing of ligand administration can bedetermined for each type of cell and disease or disorder using onlyroutine techniques.

The in vivo therapeutic methods of the invention involve direct in vivointroduction of the polynucleotides, e.g., adenoviral vector, into thecells of the subject. The polynucleotides may be introduced into thesubject systemically or locally (e.g., at the site of the disease ordisorder). Once the polynucleotides have been introduced to the subject,the ligand may be administered to induce expression of the therapeuticpolypeptide or therapeutic polynucleotide. The ligand may beadministered by any suitable method, either systemically (e.g., orally,intravenously) or locally (e.g., intraperitoneally, intrathecally,intraventricularly, direct injection into the tissue or organ where thedisease or disorder is occurring). The optimal timing of ligandadministration can be determined for each type of cell and disease ordisorder using only routine techniques.

For in vivo use, the ligands described herein may be taken up inpharmaceutically acceptable carriers, such as, for example, solutions,suspensions, tablets, capsules, ointments, elixirs, and injectablecompositions. Pharmaceutical compositions may contain from 0.01% to 99%by weight of the ligand. Compositions may be either in single ormultiple dose forms. The amount of ligand in any particularpharmaceutical composition will depend upon the effective dose, that is,the dose required to elicit the desired gene expression or suppression.

As used herein, the term “rAD.RheoIL12” refers to an adenoviralpolynucleotide vector harboring the IL-12 gene under the control of agene switch of the RheoSwitch® Therapeutic System (RTS), which iscapable of producing IL-12 protein in the presence of activating ligand.As used herein, the term “rAd.cIL12” refers to an adenoviralpolynucleotide control vector containing the IL-12 gene under thecontrol of a constitutive promoter.

As used herein, the term “IL-12p70” refers to IL-12 protein, whichnaturally has two subunits commonly referred to as p40 and p35. The termIL-12p70 encompasses fusion proteins comprising the two subunits ofIL-12 (p40 and p35), wherein the fusion protein may include linker aminoacids between subunits.

Suitable routes of administering the pharmaceutical preparations includeoral, rectal, topical (including dermal, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenous,intratumoral, intradermal, intrathecal and epidural) and by naso-gastrictube. It will be understood by those skilled in the art that the routeof administration will depend upon the condition being treated and mayvary with factors such as the condition of the recipient.

As used herein, the terms “activating” or “activate” refer to anymeasurable increase in cellular activity of a gene switch, resulting inexpression of a gene of interest.

As used herein, the terms “treating” or “treatment” of a disease referto executing a protocol, which may include administering one or moredrugs or in vitro engineered cells to a mammal (human or non-human), inan effort to alleviate signs or symptoms of the disease. Thus,“treating” or “treatment” should not necessarily be construed to requirecomplete alleviation of signs or symptoms, does not require a cure, andspecifically includes protocols that have only marginal effect on thesubject.

As used herein, the terms “in vitro engineered cells” or “in vitroengineered population of cells” or “a population of engineered cells” or“cells expressing a protein” refer to cells conditionally expressing aprotein under the control of a gene switch, which can be activated by anactivating ligand.

As used herein, the term “modified cell” refers to cells which have beenaltered by a process including, but not limited to, transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation and lipofection (lysosomefusion).

As used herein, the terms “MOI” or “Multiplicity of Infection” refer tothe average number of adenovirus particles that infect a single cell ina specific experiment (e.g., recombinant adenovirus or controladenovirus)

In another embodiment, the vector and methods of the present inventioncan be used to treat disease.

In another embodiment, the vector and methods of the present inventioncan be used to treat a kidney disease. In one embodiment, the kidneydisease is a renal failure. In another embodiment, the kidney disease ischronic renal failure.

In another embodiment, the vector and methods of the present inventioncan be used to treat anemia. In one embodiment, the anemia is anemiaassociated with kidney disease, for example, renal failure or chronicrenal failure. In another embodiment, the anemia is associated withcancer therapy with, for example, one or more chemotherapeutic agents.In another embodiment, the anemia is associated with advanced age. Inanother embodiment, the anemia is associated with impaired lungfunction. In another embodiment, the anemia is associated withmyelodisplasia. In another embodiment, the anemia is associated withradiation therapy. In another embodiment, the anemia is associated witha critical illness.

In one embodiment, the anemia is not associated with cardiac disease. Inanother embodiment, the disease, disorder or condition that isresponsive to treatment with erythropoietin is not a cardiac disease.Nonlimiting types of “cardiac disease” are congestive heart failure,hypoxia, ischemic heart disease, hypertensive heart disease, coronaryartery disease, peripheral vascular disease and ischemic cardiac events,e.g., myocardial infarction, heart attack, heart failure, arrhythmia,myocardial rupture, pericarditis, cardiogenic shock, thrombosis,embolism, atherosclerosis, and arterial stenosis.

In another embodiment, the polynucleotide comprising a polynucleotideencoding an erythropoietin or agonist thereof does not also encode withetanercept, which is a TNF receptor-Fc fusion. In another embodiment,the polynucleotide comprising a polynucleotide encoding anerythropoietin or agonist thereof is not administered to a subject towhom is also administered a polynucleotide comprising a polynucleotideencoding etanercept.

In one embodiment, the vector and methods of the present invention areused to treat multiple sclerosis. In one embodiment, the vectorcomprises a polynucleotide sequence encoding an interferon, or afragment thereof. In another embodiment, the vector comprises apolynucleotide sequence encoding an interferon-beta, or a fragmentthereof. In another embodiment, the vector comprises a polynucleotidesequence encoding myelin basic protein (MBP), or a fragment thereof. Inone embodiment, the vector comprises a polynucleotide sequence encodingan interferon, e.g., an interferon-beta, or a fragment thereof, andmyelin basic protein, or a fragment thereof.

In another embodiment, the vector and methods of the present inventionare used to treat angioedema. In another embodiment, the angioedema ishereditary angioedema. In one embodiment, the vector comprises apolynucleotide sequence encoding molecule selected from the groupconsisting of a C1 esterase inhibitor (for example, a human C1 esteraseinhibitor), a kallikrein inhibitor, and a bradykinin B2 receptorantagonist.

In another embodiment, the vector and methods of the present inventionare used to treat a disease, condition or disorder, wherein inhibitionof C1 esterase provides a therapeutically beneficial effect. In thisembodiment, the vector comprises a polynucleotide sequence encoding a C1esterase inhibitor to treat, for example, a disease, condition ordisorder selected from the group consisting of sepsis,hypercoagulability, pulmonary dysfunction, hypoxemia, hemorrhagicpancreatitis, myocardial infarction, lung transplantation, trauma,thermal injury and vascular leak.

In another embodiment, the vector and methods of the present inventionare used to treat a disease, condition or disorder wherein inhibition ofkallikrein provides a therapeutically beneficial effect. Examples ofsuch diseases, conditions or disorders include, but are not limited to,disease, conditions or disorders of the contact system. See e.g.,Shariat-Madar et al., Innate Immunity, vol. 10, no. 1, 3-13 (2004) andFrick, et al., EMBO J., (2006) 25, 5569-5578 (2006). In this embodiment,the vector comprises a polynucleotide sequence encoding a kallireininhibitor to treat, for example, a disease, condition or disorderselected from the group consisting of atherothrombosis, coronary arterydisease, Alzheimer's Disesase, inflammatory bowel disease (for example,Crohn's Disease), vascular leak, acute respiratory distress syndrome andbradykinin-mediated inflammation. In one embodiment, the vectorcomprises a polynucleotide sequence encoding a kallikrein inhibitor.Examples of kallikrein inhibitors include, but are not limited to,ecallantide and those kallikrein inhibits set forth U.S. PatentPublication Nos. 2010/0034805, 2009/0264350, 2009/0234009, 2008/0221031,2007/0213275, 2006/0264603 and 2005/0089515, each of which areincorporated by reference in their entireities.

In another embodiment, the vector and methods of the present inventionare used to treat pulmonary hypertension. In one embodiment, thepulmonary hypertension is pulmonary arterial hypertension. In anotherembodiment, the pulmonary arterial hypertension is idiopathic pulmonaryarterial hypertension. In another embodiment, the pulmonary arterialhypertension is familial pulmonary arterial hypertension. In anotherembodiment, the pulmonary arterial hypertension is pulmonary arterialhypertension associated with other diseases or conditions. In anotherembodiment, the pulmonary arterial hypertension is pulmonary arterialhypertension secondary to other conditions. In another embodiment, thepulmonary arterial hypertension is secondary pulmonary arterialhypertension. In another embodiment, the pulmonary arterial hypertensionis associated with significant venous or capillary involvement, forexample, pulmonary veno-occlusive disease and pulmonary capillaryhemangiomatosis. In another embodiment, the pulmonary arterialhypertension is persistent pulmonary hypertension of the newborn. In oneembodiment, the vector is administered intramuscularly.

In one embodiment, the term “prostaglandin synthase” is a polypeptideselected from the group consisting of prostaglandin synthase,prostaglandin synthetase, prostaglandin synthetase 1, prostaglandinsynthetase 2, prostaglandin endoperoxide synthetase, prostaglandin Esynthetase, prostaglandin H2 synthetase, prostaglandin G/H synthetase 1,prostaglandin G/H synthetase 2, PG synthetase, cyclooxygenase (COX),COX-1, COX-2 and COX-3.

The accession number for the human Prosteglandin G/H Synthase 1nucleotide sequence is NC_000009, and the accession number for the humanProstoglandin G/H Synthase 1 amino acid sequence is Accession No.:NP_000953. See, e.g., Lander et al., Nature 429: 369-374 (2004).

The accession number for the human Prosteglandin G/H Synthase 2nucleotide sequence is NC_000001, and the accession number for the humanProstoglandin G/H Synthase 2 amino acid sequence is Accession No.:NP_000954.1. See, e.g., Lander et al., Nature 431: 931-945 (2004).

The accession number for the human interferon-beta is NP_002167.1

The accession number for the human GLP-1 is RP_12738.

The accession number for the human GLP-2 is RP_10769.

The accession number for the human adiponectin is ABZ10942.1.

The accession number for the human leptin is AAH69323.1.

The accession number for the human CFTR is ABD72213.1

The accession number for the human IL-10 NP_000563.

In another embodiment, the vector and methods of the present inventionare used to treat a disease, condition or disorder wherein inhibition ofbradykinin B2 receptor provides a therapeutically beneficial effect. Inthis embodiment, the vector comprises a polynucleotide sequence encodinga bradykinin B2 receptor inhibitor to treat, for example, a disease,condition or disorder selected from the group consisting ofglomerulosclerosis, Alzheimer's Disease, cerebral edema, vascular leak,acute respiratory distress syndrome, pain, inflammation, trauma, burns,shock, allergy, and cardiovascular disease. Examples of bradykinin B2receptor inhibitors include, but are not limited to, helokinestatin andanti-bradykinin B2 receptor antibodies. The amino acid sequence ofhelokinestatin is Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Kwok, H. F.et al., Peptides 29I 65-72 (2008), which is incorporated by reference inits entirety). Nonlimiting examples of anti-bradykinin B2 receptorantibodies are set forthin Alla, S. A. et al., J. Biol. Chem. 271:1748-1755 (1996).

In one embodiment, the vector administered to the mammal afflicted withone or more of the disclosed diseases is an adenoviral vector. In oneembodiment, the vector comprises a polynucleotide encoding a geneswitch. In one aspect, the gene switch is an EcR-based gene switch. Inanother embodiment, the polynucleotide encoding a gene switch comprisesa first transcription factor sequence under the control of a firstpromoter and a second transcription factor sequence under the control ofa second promoter, wherein the proteins encoded by said firsttranscription factor sequence and said second transcription factorsequence interact to form a protein complex which functions as aligand-dependent transcription factor. In one aspect, the ligand is adiacylhydrazine. In another aspect, the ligand is selected fromRG-115819, RG-115932, and RG-115830. In yet another aspect, the ligandis an amidoketone or an oxadiazoline.

In one embodiment, a nucleic acid adenoviral vector is providedcontaining a gene switch, wherein the coding sequences for VP16-RXR andGal4-EcR are separated by the EMCV internal ribosome entry site (IRES)sequence are inserted into the adenoviral shuttle vector under thecontrol of the human ubiquitin C promoter. For example, the codingsequences for the p40 and p35 subunits of IL12 separated by an IRESsequence, and placed under the control of a synthetic induciblepromoter, are inserted upstream of the ubiquitin C promoter. In anotherexample, the coding sequence of TNF-alpha, which is placed under thecontrol of a synthetic inducible promoter, is inserted upstream of theubiquitin C promoter.

Purification of the vector to enhance the concentration can beaccomplished by any suitable method, such as by density gradientpurification (e.g., cesium chloride (CsCl)) or by chromatographytechniques (e.g., column or batch chromatography). For example, thevector of the invention can be subjected to two or three CsCl densitygradient purification steps. The vector, e.g., a replication-deficientadenoviral vector, is desirably purified from cells infected with thereplication-deficient adenoviral vector using a method that compriseslysing cells infected with adenovirus, applying the lysate to achromatography resin, eluting the adenovirus from the chromatographyresin, and collecting a fraction containing adenovirus.

In a particular embodiment, the resulting primary viral stock isamplified by re-infection of HEK 293 cells or CHO cells and is purifiedby CsCl density-gradient centrifugation.

Protein-based tags reduce or eliminate the need for highly specificpost-translational modifications for effective targeting. Usefulprotein-based tags include, but are not limited to, IGF2R targeting(IGF2 (GILT)/IGF2 engineering), transferrin receptor targeting(transferrin, TfR-targeting peptides), and Tat protein (in which cellsurface heparin sulfate proteoglycans (HSPGs) mediate internalization ofTat).

Other proteins that target to the lysosome than can be used as a taginclude, but are not limited to, Vitamin D binding protein, folatebinding protein, lactotransferrin, sex hormone binding globulin,transthyretin, pro saposin, retinol binding protein, Apo lipoprotein B,Apo lipoprotein E, prolactin, receptor associated protein (in oneembodiment, without the HNEL sequence), native transferrin, and mutanttransferring (e.g., the K225E/R651A mutant or the K225E/K553A mutant).

In one aspect, the invention provides a pharmaceutical compositionsuitable for administration to a human or a non-human comprising apopulation of in vitro engineered cells or a vector, e.g., an adenoviralvector, expressing a protein, wherein the formulation is suitable foradministration by intratumoral administration. In another embodiment, acomposition, e.g., pharmaceutical composition, comprises a vectorconditionally expressing a protein. In some embodiments, the compositioncomprises about 1×10⁵ or more particle units (pu) of the gene transfervector. A “particle unit” is a single vector particle. In certainembodiments, the composition comprises about 1×10⁶ particle units of thegene transfer vector (e.g., about 1×10⁷ or more particle units, about1×10⁸ or more particle units, or about 1×10⁹ or more particle units). Inother embodiments, the composition comprises about 1×10¹⁰ or more pu,1×10¹¹ or more pu, 1×10¹² or more pu, 1×10¹³ or more pu, 1×10¹⁴ or morepu, or 1×10¹⁵ or more pu of the gene transfer vector, especially of aviral vector, such as a replication-deficient adenoviral vector. Thenumber of particle units of the gene transfer vector in the compositioncan be determined using any suitable method known, such as by comparingthe absorbance of the composition with the absorbance of a standardsolution of gene transfer vector (i.e., a solution of known genetransfer vector concentration) as described further herein.

In one embodiment, the activating ligand is selected from the groupconsisting of RG-115819, RG-115830 and RG-115932.

The invention further provides a pharmaceutical composition comprisingan activating ligand, such as RG-115819, RG-115830 or RG-115932, whereinthe composition is suitable for administration by intraperitoneal, oral,or subcutaneous administration.

In one embodiment, the activating ligand is administered orally. Inanother embodiment, the activating ligand is administered parenterally.In another embodiment, the activating ligand is administered,intraperitoneally, subcutaneously, or intramuscularly.

A composition of the invention can further comprise a pharmaceuticallyacceptable carrier. The carrier can be any suitable carrier for the anengineered dendritic cells, gene transfer vector, or activating ligand.Suitable carriers for the composition are described in U.S. Pat. No.6,225,289. The carrier typically will be liquid, but also can be solid,or a combination of liquid and solid components. The carrier desirablyis a pharmaceutically acceptable (e.g., a physiologically orpharmacologically acceptable) carrier (e.g., excipient or diluent).Pharmaceutically acceptable carriers are well known and are readilyavailable. The choice of carrier will be determined, at least in part,by the particular components in the composition and the particularmethod used to administer the composition. The composition can furthercomprise any other suitable components, especially for enhancing thestability of the composition and/or its end-use. Accordingly, there is awide variety of suitable formulations of the composition of theinvention.

Formulations suitable for oral administration include (a) liquidsolutions, such as an effective amount of the active ingredientdissolved in diluents, such as water, saline, or orange juice, (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as solids or granules, (c) suspensions in anappropriate liquid, and (d) suitable emulsions. Tablet forms can includeone or more of lactose, mannitol, corn starch, potato starch,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatibleexcipients. Lozenge forms can comprise the active ingredient in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base (such as gelatin andglycerin, or sucrose and acacia), and emulsions, gels, and the likecontaining, in addition to the active ingredient, such excipients as areknown in the art.

Formulations suitable for administration via inhalation include aerosolformulations. The aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like. They also can be formulated as non-pressurizedpreparations, for delivery from a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnonaqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Formulations suitable for anal administration can be prepared assuppositories by mixing the active ingredient with a variety of basessuch as emulsifying bases or water-soluble bases. Formulations suitablefor vaginal administration can be presented as pessaries, tampons,creams, gels, pastes, foams, or spray formulas containing, in additionto the active ingredient, such carriers as are known in the art to beappropriate.

In addition, the composition can comprise additional therapeutic orbiologically-active agents. For example, therapeutic factors useful inthe treatment of a particular indication can be present. Factors thatcontrol inflammation, such as ibuprofen or steroids, can be part of thecomposition to reduce swelling and inflammation associated with in vivoadministration of the gene transfer vector and physiological distress.Immune system suppressors can be administered with the compositionmethod to reduce any immune response to the gene transfer vector itselfor associated with a disorder. Alternatively, immune enhancers can beincluded in the composition to upregulate the body's natural defensesagainst disease. Moreover, cytokines can be administered with thecomposition to attract immune effector cells to the tumor site.

In the particular embodiment described herein, the invention provides amethod for treating a tumor, comprising the steps in order of:

a. administering intratumorally in a mammal a population of an in vitroengineered immune cells or TSC; and

b. administering to said mammal a therapeutically effective amount of anactivating ligand.

In one embodiment, the activating ligand is administered atsubstantially the same time as the composition comprising the in vitroengineered cells or the vector, e.g., adenoviral vector, e.g., withinone hour before or after administration of the cells or the vectorcompositions. In another embodiment, the activating ligand isadministered at or less than about 24 hours after administration of thein vitro cells or the vector. In still another embodiment, theactivating ligand is administered at or less than about 48 hours afterthe in vitro engineered cells or the vector. In another embodiment, theligand is RG-115932. In another embodiment, the ligand is administeredat a dose of about 1 to 50 mg/kg/day. In another embodiment, the ligandis administered at a dose of about 30 mg/kg/day. In another embodiment,the ligand is administered daily for a period of 7 to 28 days. Inanother embodiment, the ligand is administered daily for a period of 14days. In another embodiment, about 1×10⁶ to 1×10⁸ cells areadministered. In another embodiment, about 1×10⁷ cells are administered.

The term “subject” means a mammal. Mammals include humans, rodents,monkeys, and other animals, with humans or mice being more preferred.Other mammals include veterinary animals such as dogs, cats, horses,cattle, sheep, goats, pigs and the like.

As used herein, the term “protein expression” includes withoutlimitation transcription, post-transcription, translation, and/orpost-translation.

Also included in the invention is a method of increasing mRNA or proteinexpression of a protein, comprising generating a vector conditionallyexpressing the protein, wherein said vector further comprises one ormore regulatory sequences connected to the polynucleotide sequenceencoding said protein, and adding an activating ligand, thereby inducingexpression of the protein, wherein said one or more regulatory sequencesand/or signal peptides improves expression of said protein. Variousregulatory regions for the invention including, but not limited to, 5′untranslated region (5′UTR), 3′ UTR, or both have been described. In oneembodiment, the 5′ UTR is 5U2. 5U2 is a fusion canine SERCA2 intron 2with a mutated putative consensus poly-A site, with exon 2 splice donorflanking on the 5′ end and exon 3 splice acceptor flanking on the 3′ endfollowed by a portion of the portion of bovine casein 5′UTR. In anotherembodiment, the 3′ regulatory region is a polyadenylation signal of SV40or hGH.

The invention further supports the therapeutic applications of in vitroengineered cells with conditionally expressed genes of interest asinnovative approaches for the effective and efficient treatment of humandiseases.

In this embodiment, the vector is administered to the subject withoutbeing packaged in a cell.

In one embodiment, cells are not administered intratumorally with thevector.

In another embodiment, a vector of the invention that is not containedwithin a cell is administered simulataneously with, before, or aftercells, are administered.

In one embodiment, the dosage is at least about 1.0×10⁹ viral particlesper cycle of vector administration. In another embodiment, the dosage isat least about 1.0×10¹⁰ viral particles per cycle of vectoradministration. In another embodiment, the dosage is about 1.0×10⁹ toabout 1.0×10¹³ viral particles per cycle of vector administration. Inanother embodiment, the dosage is about 1.0×10¹⁰ to about 1.0×10¹³ viralparticles per cycle of vector administration. In another embodiment, thedosage is about 1.0×10¹⁰, about 1.0×10¹¹, about 1.0×10¹² or about1.0×10¹³ viral particles per cycle of vector administration.

The activating ligand dosage is about 5-100 mg/day, e.g., about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 mg/day. In one embodiment, the activating ligand is administered atleast once a day. In another embodiment, the activating ligand isadministered once a day for about 14 days.

In one embodiment, at least two dosages of the vector (e.g., about1×10¹¹ and 1×10¹²) are used in combination with at least three differentdosage levels of the activating ligand (e.g., about 5 mg/day to about100 mg/day).

One of ordinary skill in the art will be able to optimize dosages inorder to provide range of effective plasma levels of the vector, forvarious degrees of activating ligand activation.

In one embodiment, the dosage of activating ligand administered to thesubject is changed over the period of administration of the activatingligand within the cycle of intratumoral vector administration. Inanother embodiment, the dosage of activating ligand administered to thesubject is decreased over the period of administration of the activatingligand within the cycle of intratumoral vector administration. Inanother embodiment, the dosage of activating ligand administered to thesubject is increased (escalated) over the period of administration ofthe activating ligand within the cycle of intratumoral vectoradministration.

In one embodiment, the subject is treated with 2, 3, 4, 5, 6, 7, 8, 9 or10 cycles of vector administration. In another embodiment, the subjectis treated with 3-7 cycles of vector administration. In anotherembodiment, the subject is treated with 4-6 cycles of vectoradministration. In another embodiment, the subject is treated with 5 or6 cycles of vector administration. In another embodiment, the subject istreated with 6 cycles of vector administration.

In one embodiment, each cycle of vector administration is performed 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks apart. In another embodiment, eachcycle of vector administration is performed 4 weeks apart.

In one embodiment, the dosage of the vector is changed in eachsubsequent cycle of vector administration. In another embodiment, thedosage of the vector is decreased in each subsequent cycle of vectoradministration. In another embodiment, the dosage of the vector isincreased in each subsequent cycle of vector administration.

In one embodiment, the invention also provides a pharmaceuticalcomposition comprising pharmaceutically acceptable carrier and a vectorof the invention that is not contained within a cell. Suitable carriersinclude, but are not limited to, saline, distilled water, sodiumchloride solutions, the mixtures of sodium chloride and inorganic saltsor their similar mixtures, the solutions of materials such as mannitol,lactose, dextran, and glucose, amino acid solutions such as glycine andarginine, the mixtures of organic acid solutions or salt solutions andglucose solutions, aqueous and nonaqueous, isotonic sterile injectionsolutions, which can contain antioxidants, chelating agents, buffers,bacteriostats, and solutes that render the formulation isotonic, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit dose or multidose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use.

In one embodiment, the polynucleotide of the invention is contained in ahost cell. In one embodiment, the host cell is selected from the groupconsisting of a mammalian cell, a prokaryotic cell, a bacterial cell, afungal cell, a nematode cell, an insect cell, a fish cell, a plant cell,an avian cell, a eukaryotic cell, an animal cell, a mammalian cell, aninvertebrate host cell, a vertebrate host cell, a yeast cell, azebrafish cell, a chicken cell, a hamster cell, a mouse cell, a ratcell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell,a cow cell, a pig cell, a horse cell, a sheep cell, a simian cell, amonkey cell, a chimpanzee cell, or a human cell,

In one embodiment, the host cell is not a cardiac cell or a myocyte.

Host cell transformation is well known in the art and may be achieved bya variety of methods including but not limited to electroporation, viralinfection, plasmid/vector transfection, non-viral vector mediatedtransfection, Agrobacterium-mediated transformation, particlebombardment, and the like. Expression of desired gene products involvesculturing the transformed host cells under suitable conditions andinducing expression of the transformed gene. Culture conditions and geneexpression protocols in prokaryotic and eukaryotic cells are well knownin the art. Cells may be harvested and the gene products isolatedaccording to protocols specific for the gene product.

In the event of conflict between any teaching or suggestion of anyreference cited herein and the specification, the latter shall prevail,for purposes of the invention.

All patents, patent applications and publications cited herein are fullyincorporated by reference in their entireties.

It is to be understood that the foregoing described embodiments andexemplifications are not intended to be limiting in any respect to thescope of the invention, and that the claims presented herein areintended to encompass all embodiments and exemplifications whether ornot explicitly presented herein.

U.S. application Ser. No. 12/247,738, entitled “Engineered DendriticCells And Uses For Treatment Of Cancer,” filed Oct. 8, 2008, is herebyincorporated by reference in its entirety. U.S. application Ser. No.12/241,018, entitled “Therapeutic Gene-Switch Constructs And BioreactorsFor The Expression Of Biotherapeutic Molecules, And Uses Thereof,” filedSep. 29, 2008, is also hereby incorporated by reference in its entirety.

Embodiments of the invention also include the following (where “E”indicates “Embodiment”):

E1. A method of inducing, regulating, or enhancing erythropoietin (EPO)expression in a mammal, wherein the method comprises

(a) administering an adeno-associated virus to the mammal wherein thevirus comprises a polynucleotide encoding EPO; and

(b) administering an activator ligand which induces EPO expression fromthe virus polynucleotide encoding EPO,

-   -   wherein the adeno-associated virus is administered        intramuscularly,    -   wherein the adeno-associated virus further comprises a gene        switch, wherein the gene switch comprises at least one        transcription factor sequence operably linked to a promoter,        wherein at least one transcription factor encoded by the at        least one transcription factor sequence is a ligand-dependent        transcription factor,    -   wherein the adeno-associated virus further comprises a second        promoter operably linked to the polynucleotide encoding EPO,        wherein the second promoter is activated by the at least one        ligand-dependent transcription factor following administration        of activator ligand.

E2. The method of embodiment E1, wherein the mammal is human.

E3. The method of embodiments E1 or E2, wherein expression of EPO isinduced, regulated or enhanced by controlling the administered dose ordoses of activator ligand.

E4. The method of any one of embodiments E1 to E3, wherein activatorligand is adminstered in a dose or doses sufficient to induce ormaintain EPO expression levels within a normal physiologic range.

E5. The method of any one of embodiments E1 to E4, wherein thepolynucleotide encoding EPO comprises an amino acid sequence at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:6 or SEQ ID NO: 8 (human EPO).

E6. The method of any one of embodiments E1 to E5, wherein thehematocrit or volume percentage of red blood cells in blood is increasedin the mammal.

E7. A vector comprising a polynucleotide encoding a gene switch, whereinthe polynucleotide comprises (1) at least one transcription factorsequence which is operably linked to a promoter, wherein the at leastone transcription factor sequence encodes a ligand-dependenttranscription factor, and (2) a polynucleotide encoding one or moreproteins operably linked to a promoter which is activated by theligand-dependent transcription factor, wherein the one or more proteinsis selected from the group consisting of a C1 esterase inhibitor, akallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

E8. The vector of embodiment E7, wherein one or more of the proteins isa human protein.

E9. The vector of embodiments E7 or E8, wherein the vector is a viralvector.

E10. The vector of embodiment E9, wherein the viral vector is selectedfrom the group consisting of an adenovirus, an adeno-associated virus, aretrovirus, a pox virus, a baculovirus, a vaccinia virus, a herpessimplex virus, an Epstein-Barr virus, a geminivirus, a pseudorabiesvirus, a parvovirus, and a caulimovirus virus vector.

E11. The vector of any one of embodiments E7 to E10, wherein the geneswitch is an ecdysone receptor (EcR)-based gene switch.

E12. The vector of any one of embodiments E7 to E11, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence under the control of a first promoter and a secondtranscription factor sequence under the control of a second promoter,wherein a first transcription factor encoded by the first transcriptionfactor sequence and a second transcription factor encoded by the secondtranscription factor sequence interact to form a complex which functionsas a ligand-dependent transcription factor.

E13. The vector of any one of embodiments E7 to E11, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence and a second transcription factor sequence under thecontrol of a promoter, wherein a first transcription factor encoded bythe first transcription factor sequence and a second transcriptionfactor encoded by the second transcription factor sequence interact toform a complex which functions as a ligand-dependent transcriptionfactor.

E14. The vector of any one of embodiments E7 to E13, wherein the firsttranscription factor sequence and the second transcription factorsequence are connected by an EMCV internal ribosomal entry site (IRES).

E15. The vector of any one of embodiments E7 to E14, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acidencoded by SEQ ID NO: 9 (human myelin basic protein).

E16. The vector of any one of embodiments E7 to E15, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 (humanC1 esterase inhibitor.).

E17. The vector of any one of embodiments E7 to E16, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11(ecallantide).

E18. The vector of any one of embodiments E7 to E17, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 (prostaglandin synthase).

E19. The vector of any one of embodiments E7 to E17, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17 (GLP-1)or SEQ ID NO: 18 (GLP-2).

E20. The vector of any one of embodiments E7 to E17, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19(Adiponectin).

E21. The vector of any one of embodiments E7 to E17, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20(Leptin).

E22. The vector of any one of embodiments E7 to E17, wherein one of theone or more proteins comprises an amino acid sequence at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21 (CFTR).

E23. A method of producing a population of cells expressing one or moreproteins, wherein the method comprises modifying the cells with arecombinant vector conditionally expressing one or more proteins,wherein the vector comprises a polynucleotide encoding a gene switch,wherein the polynucleotide comprises (1) at least one transcriptionfactor sequence operably linked to a promoter, wherein the at least onetranscription factor sequence encodes a ligand-dependent transcriptionfactor, and (2) a polynucleotide encoding one or more proteins linked toa promoter which is activated by the ligand-dependent transcriptionfactor, wherein the one or more proteins are selected from the groupconsisting of a C1 esterase inhibitor, a kallikrein inhibitor, abradykinin B2 receptor inhibitor, a prostaglandin synthase, aglucagon-like peptide-1 (GLP-1), a glucagon-like peptide-2 (GLP-2),adiponectin, leptin, and cystic fibrosis transmembrane conductanceregulator (CFTR).

E24. The method of embodiment E23, wherein one or more of the proteinsis a human protein.

E25. The method of embodiments E23 or E24, wherein the vector is a viralvector.

E26. The method of embodiment E25, wherein the viral vector is selectedfrom the group consisting of an adenovirus, an adeno-associated virus, aretrovirus, a pox virus, a baculovirus, a vaccinia virus, a herpessimplex virus, an Epstein-Barr virus, a geminivirus, a pseudorabiesvirus, a parvovirus, and a caulimovirus virus vector.

E27. The method of any one of embodiments E23 to E26, wherein the geneswitch is an ecdysone receptor (EcR)-based gene switch.

E28. The method of any one of embodiments E23 to E27, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence under the control of a first promoter and a secondtranscription factor sequence under the control of a second promoter,wherein a first transcription factor encoded by the first transcriptionfactor sequence and a second transcription factor encoded by the secondtranscription factor sequence interact to form a complex which functionsas a ligand-dependent transcription factor.

E29. The method of any one of embodiments E23 to E27, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence and a second transcription factor sequence under thecontrol of a promoter, wherein a first transcription factor encoded bythe first transcription factor sequence and a second transcriptionfactor encoded by the second transcription factor sequence interact toform a complex which functions as a ligand-dependent transcriptionfactor.

E30. The method of embodiments E29, wherein the first transcriptionfactor sequence and the second transcription factor sequence areconnected by an EMCV internal ribosomal entry site (IRES).

E31. A population of cells which have been modified with a recombinantvector conditionally expressing one or more proteins, wherein the vectorcomprises a polynucleotide encoding a gene switch, wherein thepolynucleotide comprises (1) at least one transcription factor sequenceoperably linked to a promoter, wherein the at least one transcriptionfactor sequence encodes a ligand-dependent transcription factor, and (2)a polynucleotide encoding one or more proteins selected from the groupconsisting of a C1 esterase inhibitor, a kallikrein inhibitor, abradykinin B2 receptor inhibitor, a prostaglandin synthase, aglucagon-like peptide-1 (GLP-1), a glucagon-like peptide-2 (GLP-2),adiponectin, leptin, and cystic fibrosis transmembrane conductanceregulator (CFTR).

E32. The population of cells of embodiment E31, wherein one or more ofthe proteins is a human protein.

E33. The population of cells of embodiments E31 or E32, wherein thevector is a viral vector.

E34. The population of cells of embodiment E33, wherein the viral vectoris selected from the group consisting of an adenovirus, anadeno-associated virus, a retrovirus, a pox virus, a baculovirus, avaccinia virus, a herpes simplex virus, an Epstein-Barr virus, ageminivirus, a pseudorabies virus, a parvovirus, and a caulimovirusvirus vector.

E35. The population of cells of any one of embodiments E31 to E34,wherein the gene switch is an ecdysone receptor (EcR)-based gene switch.

E36. The population of cells of any one of embodiments E31 to E35,wherein the polynucleotide encoding a gene switch comprises a firsttranscription factor sequence under the control of a first promoter anda second transcription factor sequence under the control of a secondpromoter, wherein a first transcription factor encoded by the firsttranscription factor sequence and a second transcription factor encodedby the second transcription factor sequence interact to form a complexwhich functions as a ligand-dependent transcription factor.

E37. The population of cells of any one of embodiments E31 to E35,wherein the polynucleotide encoding a gene switch comprises a firsttranscription factor sequence and a second transcription factor sequenceunder the control of a promoter, wherein a first transcription factorencoded by the first transcription factor sequence and a secondtranscription factor encoded by the second transcription factor sequenceinteract to form a complex which functions as a ligand-dependenttranscription factor.

E38. The population of embodiments E37, wherein the first transcriptionfactor sequence and the second transcription factor sequence areconnected by an EMCV internal ribosomal entry site (IRES).

E39. A method for treating a disease in a mammal, comprising:

(a) administering a population of cells which conditionally express oneor more proteins; and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands;

thereby inducing expression of the one or more proteins, wherein the oneor more proteins is selected from the group consisting of a C1 esteraseinhibitor, a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).

E40. A method for treating a disease in a mammal, comprising:

(a) administering to the mammal a vector for conditionally expressingone or more proteins, the vector comprising a polynucleotide encoding agene switch, wherein the polynucleotide comprises

(1) at least one transcription factor sequence which is operably linkedto a promoter, wherein the at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and

(2) a polynucleotide encoding one or more proteins operably linked to apromoter which is activated by the ligand-dependent transcriptionfactor, and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands; thereby inducing expression of the oneor more proteins and treating the disease,

wherein the one or more proteins is selected from the group consistingof a C1 esterase inhibitor, a kallikrein inhibitor, a bradykinin B2receptor inhibitor, a prostaglandin synthase, a glucagon-like peptide-1(GLP-1), a glucagon-like peptide-2 (GLP-2), adiponectin, leptin, andcystic fibrosis transmembrane conductance regulator (CFTR).

E41. The method of embodiments E39 or E40, wherein at least one of theproteins is a C1 esterase inhibitor and the disease is selected from thegroup consisting of angioedema, hereditary angioedema, sepsis,hypercoagulability, pulmonary dysfunction, hypoxemia, hemorrhagicpancreatitis, myocardial infarction, lung transplantation, trauma,thermal injury, or vascular leak.

E42. The method of embodiments E39 or E40, wherein at least one of theproteins is a kallikrein inhibitor and the disease is selected from thegroup consisting of angioedema, hereditary angioedema, atherothrombosis,coronary artery disease, Alzheimer's Disease, inflammatory boweldisease, Crohn's Disease, vascular leak, acute respiratory distresssyndrome, bradykinin-mediated inflammation and a disease, condition ordisorders of the contact system.

E43. The method of embodiments E39 or E40, wherein at least one of theproteins is a bradykinin B2 receptor inhibitor and the disease isselected from the group consisting of angioedema, hereditary angioedema,bradykinin-mediated inflammation, glomerulosclerosis, Alzheimer'sDisease, cerebral edema, vascular leak, acute respiratory distresssyndrome, pain, inflammation, trauma, burns, shock, allergy, andcardiovascular disease.

E44. The method of embodiments E39 or E40, wherein at least one of theproteins is a prostaglandin synthase and the disease is selected fromthe group consisting of pulmonary hypertension, pulmonary arterialhypertension (PAH), idiopathic pulmonary arterial hypertension, familialpulmonary arterial hypertension, secondary pulmonary arterialhypertension, pulmonary veno-occlusive disease, pulmonary capillaryhemangiomatosis, persistent pulmonary hypertension of the newborn.

E45. The method of embodiments E39 or E40, wherein at least one of theproteins is a glucagon-like peptide-1 (GLP-1) and the disease isdiabetes or other metabolic disease or disorder.

E46. The method of embodiments E39 or E40, wherein at least one of theproteins is a glucagon-like peptide-2 (GLP-2) and the disease isdiabetes or other metabolic disease or disorder.

E47. The method of embodiments E39 or E40, wherein at least one of theproteins is adiponectin and the disease is diabetes or other metabolicdisease or disorder.

E48. The method of embodiments E39 or E40, wherein at least one of theproteins is leptin and the disease is diabetes or other metabolicdisease or disorder.

E49. The method of embodiments E39 or E40, wherein at least one of theproteins is cystic fibrosis transmembrane conductance regulator (CFTR)and the disease is cystic fibrosis.

E50. A method for treating multiple sclerosis in a mammal, comprising:

(a) administering to the mammal a vector for conditionally expressingone or more proteins, the vector comprising a polynucleotide encoding agene switch, wherein the polynucleotide comprises

(1) at least one transcription factor sequence which is operably linkedto a promoter, wherein the at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and

(2) a polynucleotide encoding one or more proteins operably linked to apromoter which is activated by the ligand-dependent transcriptionfactor, and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands; thereby inducing expression of the oneor more proteins and treating the disease,

wherein the one or more proteins is selected from the group consistingof myelin basic protein (MBP) and interferon-beta (IFN-B).

E51. The method of embodiment E50, wherein one of the one or moreproteins comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 (human myelinbasic protein).

E52. The method of embodiment E50, wherein one of the one or moreproteins comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to to SEQ ID NO: 17(interferon-beta).

E53. The method of embodiment E50, wherein the one or more proteinscomprise both myelin basic protein (MBP) and interferon-beta (IFN-B).

E54. The method of any one of embodiments E50 to E53, wherein one ormore of the proteins is a human protein.

E55. The method of any one of embodiments E50 to E54, wherein the vectoris a viral vector.

E56. The method of embodiments E55, wherein the viral vector is selectedfrom the group consisting of an adenovirus, an adeno-associated virus, aretrovirus, a pox virus, a baculovirus, a vaccinia virus, a herpessimplex virus, an Epstein-Barr virus, a geminivirus, a pseudorabiesvirus, a parvovirus, and a caulimovirus virus vector.

E57. The method of any one of embodiments E50 to E56, wherein the geneswitch is an ecdysone receptor (EcR)-based gene switch.

E58. The method of any one of embodiments E50 to E57, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence under the control of a first promoter and a secondtranscription factor sequence under the control of a second promoter,wherein a first transcription factor encoded by the first transcriptionfactor sequence and a second transcription factor encoded by the secondtranscription factor sequence interact to form a complex which functionsas a ligand-dependent transcription factor.

E59. The method of any one of embodiments E50 to E57, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence and a second transcription factor sequence under thecontrol of a promoter, wherein a first transcription factor encoded bythe first transcription factor sequence and a second transcriptionfactor encoded by the second transcription factor sequence interact toform a complex which functions as a ligand-dependent transcriptionfactor.

E60. The method of embodiment E59, wherein the first transcriptionfactor sequence and the second transcription factor sequence areconnected by an EMCV internal ribosomal entry site (IRES).

E61. A method for treating inflammatory bowel or Crohn's disease in amammal, comprising:

(a) administering to the mammal a vector for conditionally expressingone or more proteins, the vector comprising a polynucleotide encoding agene switch, wherein the polynucleotide comprises

(1) at least one transcription factor sequence which is operably linkedto a promoter, wherein the at least one transcription factor sequenceencodes a ligand-dependent transcription factor, and

(2) a polynucleotide encoding one or more proteins operably linked to apromoter which is activated by the ligand-dependent transcriptionfactor, and

(b) administering to the mammal a therapeutically effective amount ofone or more activating ligands; thereby inducing expression of the oneor more proteins and treating the disease,

wherein one of the one or more proteins is interleukin-10 (IL-10).

E62. The method of embodiment E61, wherein one of the one or moreproteins comprises an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22 (interleukin-10).

E63. The method of embodiments E61 or E62, wherein the interleukin-10 isa human IL-10 protein.

E64. The method of any one of embodiments E61 to E63, wherein the vectoris a viral vector.

E65. The method of embodiments E64, wherein the viral vector is selectedfrom the group consisting of an adenovirus, an adeno-associated virus, aretrovirus, a pox virus, a baculovirus, a vaccinia virus, a herpessimplex virus, an Epstein-Barr virus, a geminivirus, a pseudorabiesvirus, a parvovirus, and a caulimovirus virus vector.

E66. The method of any one of embodiments E61 to E65, wherein the geneswitch is an ecdysone receptor (EcR)-based gene switch.

E67. The method of any one of embodiments E61 to E66, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence under the control of a first promoter and a secondtranscription factor sequence under the control of a second promoter,wherein a first transcription factor encoded by the first transcriptionfactor sequence and a second transcription factor encoded by the secondtranscription factor sequence interact to form a complex which functionsas a ligand-dependent transcription factor.

E68. The method of any one of embodiments E61 to E66, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence and a second transcription factor sequence under thecontrol of a promoter, wherein a first transcription factor encoded bythe first transcription factor sequence and a second transcriptionfactor encoded by the second transcription factor sequence interact toform a complex which functions as a ligand-dependent transcriptionfactor.

E69. The method of embodiment E68, wherein the first transcriptionfactor sequence and the second transcription factor sequence areconnected by an EMCV internal ribosomal entry site (IRES).

E70. A composition comprising the vector of any one of embodiments E7 toE22, or the population of cells of any one of E31 to E38, and apharmaceutically acceptable carrier.

E71. The composition of embodiments E70, which is administeredsystemically, intravenously, intratumorally, orally, intraperitoneally,intramuscularly, intravertebrally, intracerebrally, intrathecally,intradermally, or subcutaneously.

E72. A medicament comprising the vector of any one of embodiments E7 toE22, or the population of cells of any one of E31 to E38, and apharmaceutically acceptable carrier.

E73. The medicament of embodiment E72, which is administeredsystemically, intravenously, intratumorally, orally, intraperitoneally,intramuscularly, intravertebrally, intracerebrally, intrathecally,intradermally, or subcutaneously.

E74. A kit comprising the vector of any one of embodiments E7 to E22 orthe population of cells of any one of embodiments E31 to E38.

E75. The vector of any one of embodiments E7 to E22, or the populationof cells of any one of embodiments E31 to E38, and a ligand whichactivates the gene switch.

E76. The vector or population of cells and ligand of embodiments E75,wherein the ligand is a diacylhydrazine.

E77. The vector or population of cells and diacylhydrazine ligand ofembodiments E76, wherein the diacylhydrazine is RG-115819, RG-115830 orRG-115932.

E78. The vector or population of cells and ligand of embodiments E75,wherein the ligand is an amidoketone or oxadiazoline.

E79. The vector of any one of embodiments E7 to E22, the method of anyone of embodiments E23 to E30 and embodiments E39 to E69, the populationof cells of any one of embodiments E31 to E38, wherein the ligand whichactivates ligand-dependent transcription is a diacylhydrazine.

E80. The vector, method, or population of cells of embodiments E79,wherein the diacylhydrazine is RG-115819, RG-115830 or RG-115932.

E81. The vector of any one of embodiments E7 to E22, the method of anyone of embodiments E23 to E30 and E39 to E69, the population of any oneof embodiments E31 to E38, wherein the ligand which activatesligand-dependent transcription is an amidoketone or oxadiazoline.

E82. A kit comprising the vector of any one of embodiments E7 to E22, orthe population of cells of any one of embodiments E31 to E38, and aligand.

E83. The kit of embodiment E82, wherein the ligand is a diacylhydrazine.

E84. The kit of embodiment E83, wherein the diacylhydrazine isRG-115819, RG-115830 or RG-115932.

E85. The kit and ligand of embodiment E82, wherein the ligand is anamidoketone or oxadiazoline.

Example 1 Effect of Local Injection of Ad-RTS-mIL12 Against Local andContralateral Tumors in the B16F0 Melanoma Model

We investigated if the treatment of a local tumor with Ad-RTS-mIL12(resulting in local tumor growth regression) would also lead toanti-tumor activity in the distant tumor. Toward this goal, we developedmelanoma tumors on both flank regions of immunocompetent mice (C57BL/6)and treated the tumor on the right flank with Ad-RTS-mIL12 in thepresence of activator ligand.

Six- to eight-week-old female C57Bl/6 mice were purchased from Harlan(USA). Animal care and experimental procedures were performed accordingto Intrexon's Institutional Animal Care and Use Committee guidelines.

The murine melanoma (B16F0) cells were purchased from ATCC (Manassas,Va.). B16F0 cells were grown in Dulbecco's modified Eagle's medium(ATCC, Manassas, Va.). The DMEM was supplemented with heat-inactivatedfetal calf serum (FCS) 10% v/v, 2-mM L-glutamine (Atlanta Biologicals,Inc, Lawrenceville, Ga.), 100 IU/ml penicillin G, and 100 μg/mlstreptomycin. The cells were grown at 37° C. in 5% CO2. All cell lineswere routinely tested and found to be free of mycoplasma.

A total of 45 C57BL/6 animals were inoculated subcutaneously with murinemelanoma, B16F0 (ATCC), 1e5 cells/50 ul, on the right and left hindflanks. Twelve days later, when macroscopic tumor was visible, theanimals were randomized into four groups of ten animals, as shown in theTable below: No treatment (Group 1), Activator alone (Group 2),Ad-RTS-mIL2 alone (1×10¹⁰ vp dose, Group 3), and Ad-RTS-mIL2 (1×10¹⁰ vpdose) with activator ligand present in the chow (1000 mg/kg chow, Groups4).

Cohorts receiving activator ligand were fed rodent chow blended with theactivator ligand RG-115932 ad libitum until the end of the study.Cohorts not receiving activator continued to receive a regular diet.

Vector was administered intratumorally on Day 12 and Day 19 post tumorcell inoculation. Tumor size and body weight of each mouse weremonitored three times a week until the end of the study. The animalswere sacrificed when their cumulative tumor size exceeded 1000 mm³ ordisplayed body weight loss >15% for greater than 3 days. Upon completionof the study, all remaining animals were euthanized.

The tumor volumes were calculated using the formula, L×W²/2. Tumor sizesare shown as mean±SE. Statistical analysis was performed usingatwo-tailed t test. Differences between groups were consideredsignificant when p<0.05.

Treatment Tumor Treatment Days post cell size, body Groups N Chow (1e10vp) inoculation weight 1 10 Normal Mon, Wed and Fri 2 10 Custom Mon, Wed(1000 mg/kg) and Fri 3 10 Normal Ad-RTS- Day 12, 19 Mon, Wed mIL-12 andFri 4 10 Custom Ad-RTS- Day 12, 19 Mon, Wed (1000 mg/kg) mIL-12 and Fri

When the tumor on the right flank reached an average volume of 44 mm³,treatment was initiated. For localized delivery, two intratumoralinjections were given into right tumors with 1e10 vp of Ad-RTS-mIL12 7days apart. These animals received activator ligand chow (1000 mg/kg).The control animals received either no treatment or activator ligand(1000 mg/kg) alone or Ad-RTS-mIL12 without activator. Twenty four hoursprior to vector administration, the indicated cohorts received activatorligand (1000 mg/kg). These animals thrice weekly for any signs of tumorprogression or regression. As shown in FIG. 1A the control animalstreated with either activator ligand or no treatment had average tumorvolumes of 924±80 mm³ and 884±142 mm³, respectively on day 21 post cellinoculation.

In contrast, the tumors treated with Ad-RTS-mIL12+activator ligand hadtumor volumes of 86±138 mm³ thereby indicating a statisticallysignificant (p<0.0001) ˜91% tumor growth inhibition compared to animalswith no treatment. Ad-RTS-mIL12 without activator ligand had tumorvolume 565±305 mm³ and was not significant (p<0.07) relative to controlanimals. These data demonstrated that Ad-RTS-mIL12 without activatorligand had little impact on tumor growth while Ad-RTS-mIL12 in thepresence of activator ligand had marked anti-tumor activity.

Importantly, the animals that received Ad-RTS-mIL12 plus ligand into theright flank tumor, displayed a statistically significant (p<0.005) tumorgrowth inhibition of 81% of the uninjected left flank tumor (189±231mm³) as compared to the uninjected contralateral tumors (1019±233 mm³)in the control group with no treatment (FIG. 1B). However, forAd-RTS-mIL12 without ligand-treated tumors, there was no significantreduction in the contralateral untreated tumors. The tumors in thecontrol group treated with either Ad-RTS-mIL12 alone or activator ligandalone, as well as the contralateral uninjected tumors, continued to growmuch more rapidly after 21 days post-tumor implantation. These datasuggest that the systemic immune response against tumor cells thatdeveloped following treatment with Ad-RTS-mIL12 plus ligand in theprimary tumor may be responsible for the anti-tumor effect observed inthe distant untreated tumor.

Body weight was measured as a function of toxicity in this study.Animals were weighed three times a week until completion of the study.The mice were observed frequently for overt signs of any adverse andtreatment related side effects. Acceptable toxicity was defined as amean body-weight loss of less than 15% during the study. All treatmentsin this study were well tolerated. Maximum mean body weight losses werewithin acceptable limits (<15%) (FIG. 2) and no treatment related deathswere found. However, we found sporadic death in all the groups.

It has been well established that the immune system is capable ofrecognizing tumor-specific antigens and of eradicating malignant cells(Brunda, M. J. et al., J. Exp. Med. 178: 1223-1230 (1993); Brunda, M. J.et al., Cancer Chemother. Pharmacol. 38 (Suppl): S16-S21 (1996); andGolab J. et al., Int. J. Mol. Med. 3(5):537-44 (1999)). However, theability to harness the immune system for therapeutic purposes in cancertreatment remains elusive. This strategy involves the use ofintratumoral injection of replication defective virus vectors withregulated IL-12 expression, Ad-RTS-mIL12, to reduce the tumor growthwithout inducing systemic toxicity (Komita, H. et al, Cancer Gene Ther.16: 883-91 (2009)) and to activate the immune system to kill distal andmetastatic cancers.

In this study, intratumoral injection of established tumors withAd-RTS-mIL12 in the presence of activator ligand inhibited tumor growthin B16F0 model. Here, using a bilateral established subcutaneous B16F0tumor model, in C57BL/6 mice, it was demonstrated that unilateralintratumoral injection with Ad-RTS-mIL12 caused a significant reductionin the growth of both the injected and contralateral uninjected tumors.This antitumor effect was significant compared to animals treated withactivator ligand alone or Ad-RTS-mIL12 without activator ligand. Theseresults suggests that direct delivery of Ad-RTS-mIL12 into the tumormicroenvironment provides a therapeutic benefit and generates protectiveanti-tumor immunity against metastatic cancer cells.

Example 2 IL-12 and IFNalpha Combination Therapy

Current cancer immunotherapies have provided limited success in theclinic and innovative strategies are required to further enhance theeffectiveness of an anti-tumor immune response. This study assessedanti-tumor activity utilizing intratumoral (i.t.) administration ofadenovirus (Ad) with the novel Rheoswitch Therapeutic System® (RTS®), aninducible promoter system, for regulated expression of murine IL-12 orIFNa. Oral administration of a small molecule activator ligand (AL),INXN-1001, regulates the expression of IL-12 in Ad-RTS-mIL-12 and IFNain Ad-RTS-mIFNa. The regulated expression and therapeutic benefit ofIL-12 and IFNa from i.t. administered Ad-RTS-mIL-12 and Ad-RTS-mIFNa,either alone or in combination, was examined in syngeneic Lewis lungcarcinoma (LLC) and syngeneic mammary carcinoma (4T1) models.

In the LLC model, daily treatment with oral AL alone (administered infeed at 1000 ppm, representing a daily dose of ˜225 mg/kg/day) or i.t.cytokine gene therapy in the absence of AL did not result in significantinhibition of tumor growth compared to control, untreated tumors. Incontrast, i.t. injection with 10¹⁰ vp Ad-RTS-mIL-12 or Ad-RTS-mIFNa anddaily oral AL led to significant tumor growth inhibition by day 25 (72and 71%, respectively; p<0.05). Notably, combined treatment of the LLCtumors with Ad-RTS-mIL-12 and Ad-RTS-mIFNa with oral AL resulted insignificant anti-tumor effect compared to either treatment alone (97%growth inhibition; p<0.05) without overt toxicity as assessed by nochange in body weight. In the 4T1 model, i.t. treatment with 10¹⁰ vp ofAd-RTS-mIL-12 plus AL or Ad-RTS-mIFNa plus AL led to 58 and 53%inhibition of tumor growth compared to control untreated tumors by day34 (p<0.05). Notably, concomitant treatment with both Ad-RTS-mIL-12 plusAL and Ad-RTS-mIFNa plus AL resulted in enhanced anti-tumor activitywith 80% growth inhibition. These data indicate that the combinedtreatment strategy using RTS-regulated IL-12 and IFNa in Ad vectorsconcomitant with AL induces effective therapeutic activity againstaggressive murine tumors. Future studies will investigate the mechanismby which both IL-12 and IFNα exert their anti-tumor effect in the abovetumor models.

Interleukin-12 (IL-12) is a potent pleiotropic cytokine used fortreatment of several infectious and malignant diseases. IL-12 antitumoractivity is mediated by direct tumor cell cytotoxicity, anti-angiogenicproperties and enhancement of immunoregulatory activities includingactivation of natural killer cells, CD4⁺ T cells and CD8⁺ T cells.Despite these anti-tumor effects, systemic infusion of recombinant IL-12in humans results in severe systemic toxicity which severely limits itsuse clinically.

Interferon alpha (IFNa) is a cytokine with potent antiviral andantitumor effects. Administration of IFNa stimulates T cells and naturalkiller cells proliferation leading to tumor cell cytotoxicity,anti-angiogensis, and increased expression of major histocompatibilitycomplex (MHC), tumor antigens as well as adhesion molecules. Similarlyto IL-12, high levels of IFNa also displays severe side effectsincluding influenza-like syndrome, severe nausea, fatigue, anddepression.

Therefore, there is a clear need to control the expression levels ofthese cytokines. The RTS™ (Rheoswitch Therapeutic System) represents anovel regulation system that allows control of gene expression usingINXN-1001, the activator ligand which is an orally bioavailable smallmolecule drug. Utilizing the RTS technology to control cytokineexpression, we have previously demonstrated tumor growth reduction inseveral preclinical animal models using either Ad-RTS-IL-12 transduceddendritic cells and more recently, the direct intratumoral (IT)injection of Ad-RTS-IL-12. This has led to the initiation of a Phase 1clinical trial of direct Ad-RTS-IL-12 into tumor lesions of patientswith Stage III/IV malignant melanoma.

Combinatorial therapies are showing promising potential in preclinicalmodels as well as in the clinic. Therefore, in this study, we addressthe synergistic effect of Ad-RTS-IL-12 and Ad-RTS-IFNa co-administeredIT in two different syngeneic tumor models, Lewis Lung carcinoma (LLC)and 4T1 breast cancer.

The RheoSwitch Therapeutic System (RTS) contains three basic components:(1) an inducible promoter, (2) a ligand-inducible transcription factorand a co-activation partner (3) RheoSwitch activator ligand (AL).

In the absence of ligand, the switch protein complex provides an “off”signal. In contrast, in the presence of ligand, the complex changesconformation and provides a dose-dependent “on” signal for target geneexpression. In vivo, the orally administered AL turns on gene expressionwithin 24 hours, and upon withdrawal of the AL, gene expression returnsto baseline levels within about 24 hours.

LLC and 4T1 cells were transiently transduced with Ad-RTS-murine IL12 orAd-RTS-murine IFNa at a MOI of 100. To induce gene expression, activatorligand INXN-1001 was added to the culture medium at a concentration of75 nM or treated with 0.1% DMSO as a control. Supernatants werecollected at 48 h and cytokine levels assessed by ELISA. n=3, mean±s.d.The results are shown in FIG. 3.

LLC and 4T1 cells were transiently transduced with Ad-RTS-murine IL12 orAd-RTS-murine IFNa at a MOI of 100. To induce gene expression, activatorligand INXN-1001 was added to the culture medium at a concentration of75 nM or treated with 0.1% DMSO as a control. Supernatants werecollected at 48 h and cytokine levels assessed by ELISA. n=3, mean±s.d.The results are shown in Figure. Intratumor administration of adenoviruson specific days are indicated by arrows in FIG. 4. Controls includedPBS and Ad-RTS-Luc injected mice treated with or without activatorligand (INXN-1001), which were grouped since no effects on tumors sizeoccurred. Percentage of tumor inhibition is reported in the graph andare significantly lower than controls and single cytokine therapy groups(p<0.01). n=5, mean±s.e.m.

In a separate study, we injected mice with 1×10⁵ 4T1 cancer cells vias.c. route. When tumors reached palpable size, mice were randomized intotreatment groups with Ad-RTS-IL-12 and/or Ad-RTS-IFNa (10¹⁰vp/mouse/vector) alone or in combination administered intratumorally.Sera and tumors were collected at 48 hours after intratumor injection.Controls includes PBS and Ad-RTS-Luc injected mice with or withoutINXN-1001 activator ligand which were grouped since no effects on tumorsize occurred. INXN-1001 was formulated in Labrasol and delivered byoral gavage on a daily basis. Cytokines levels in circulation andintra-tumor were assessed using sera and tumor homogenates and detectedby ELISAs. n=4, mean±s.e.m. The results are shown in FIG. 5.

LLC and 4T1 (results later this week) cells were transduced withAd-RTS-IL-12 and Ad-RTS-IFNa alone or in combinations at concentrationof 500 MOI for each vector. Transduced cells were cultured in thepresence of 75 nm INXN-1001 or 0.1% DMSO for 48 h. Cell culturesupernatants were collected for cytokine analysis. Cells were alsoharvested for flow cytometric analysis of MHC Class I expression. n=2-3,mean±s.d. The results are shown in FIG. 6.

The data demonstrate a synergistic effect of IL12 and IFNa cytokines toinhibit tumor growth. While IL-12 was previously shown to reduce tumorgrowth, the combination with Ad-RTS-IFNa significantly enhancedefficacy, as measured by tumor growth reduction in the LLC and 4T1models. Notably, no toxicity was observed after cytokines expression,demonstrating the regulated expression of IL12 and IFNa with theRheoswitch system. Mechanistically, IFNa triggers MHC-I expression ontumor cells, thus leading to an augmented cell death. Additional MOAstudies are currently underway.

Overall, cytokines combination delivered in a safe, controlled andinducible fashion represent a novel strategy to treat aggressive tumorsthat commonly affect human population.

Example 3 Methods

The experiments in the following Examples 4-8 were performed as follows.

Animals

Female C3H/H and Balb/c mice, 6-8 weeks old, were purchased from HarlanLaboratories. Animals were maintained and treated in accordance with theInstitutional Animal Care and Use Committee of Intrexon Corporation.Animals were fed water and alfalfa free chow with 18% protein purchasedfrom Harlan.

All procedures were performed on anaesthetized animals. Area surroundingthe quadriceps muscle was pre-injected with 50 U (in 50 μl final volume)of Hylanex (rHuPH20, Halozyme) 1 hr prior to DNA injection. A smallincision was made to expose the quadriceps muscle and 250 ug ofpre-clinical grade RTS-hEPO plasmid was injected in a final volume of100 ul using a tuberculin syringe fitted with a 29 gauge needle. Theincision was quickly sutured. The DNA was electroporated with the helpof a 2-needle electrode (5 mm) inserted into the muscle, with eachneedle on either side of the DNA injection site. 8 pulses at 50 V/cm (20ms, 1 Hz) were delivered to enhance gene transfer.

Ligand

RG-115932 ligand was formulated into animal diet and was given at aconcentration of 1000 mg/kg. For OG ligand was formulated in Labrasol at10 mg/ml.

Intramuscular Administration of AAV-HuEPO (0034A, 0034B)

Animals were anesthetized, their quadriceps were visualized and theinjection site sterilized. The mice were injected with vector using a0.5 ml insulin syringe and a 29.5 gauge needle. Each animal was injectedwith total of 10¹¹ virus genomes in a 100 ul volume. After theprocedure, the animals were placed in their cage and observed for normalambulation.

Protein Analyses

Plasma samples were assayed for the presence of human erythropoietinusing Enzyme Linked Immunosorbent Assay (ELISA) (StemCell Technologies,#01630).

Hematocrit Measurement

Mice were bled via retro-orbital sinus and samples were measured byHeska CBC hematology analyzer.

Example 4 Evaluation of Human Erythropoietin Efficacy in Mice FollowingSingle Intramuscular Administration of AAV-RTS-HuEpo

“AAV-RTS-HuEPO” refers to the adeno-assocated viral vector-RheoSwitch®Therapeutic System-Human EPO. The nucleic acid sequence of the signalpeptide sequence, human erythropoietin sequence, and stop codon are setforth in SEQ ID NO: 8.

The goal of this study was to determine if AAV-mediated, intramuscular(IM) delivery of the HuEPO transgene to mice would result in measurablehuEPO expression and a concurrent increase in hematocrit (HCT). TwoAAV-huEPO vectors, 0034A and 0034B (FIGS. 7A and 7B), were tested usingtwo mouse strains, C3H/H and BALB/c. The huEPO expression cassettes inboth AAV vectors are under the control of the inducible promoter. HuEpolevels in the plasma and HCT were measured weekly.

On the start of the study, mice were bled for baseline hematocritlevels, followed by IM administration of 10¹¹ AAV-HuEPO viral genomesper animal. Animals received activator ligand-containing chow (18-1000)starting on the day of AAV administration. Control animals receivedeither vector administration and normal chow (no activator ligand), orthey received vehicle-alone IM administration (saline) with or withoutactivator ligand. HCTs were measured in all groups every 7 days. Bothgroups that received the HuEPO vectors in the presence of activatorligand displayed elevated hematocrits.

As shown in FIG. 8, 0034A displayed a 40% HCT increase and and 0034B,displayed a 25% HCT increase, compared to the controls. On study day 29,ligand inducer was removed and all groups received normal chow.Importantly, hematocrit levels decreased into the normal range by twoweeks after removal of the activator ligand. Ligand-containing chow wasreintroduced on day 50, again resulting in an increased HCT and removedagain on day 64, resulting in a decrease in HCT.

These data demonstrate regulated HuEPO expression following a single IMadministration of the AAV-huEPO vector that mediated physiologicalchanges. In the presence of activator ligand, high levels of HuEPO wereexpressed with resulted in an increase in HCT. When activator ligand wasremoved, HCT fell within the normal range. HuEPO expression andsubsequent HCT increases could be induced at least twice by introductionof the activator ligand.

Example 5 Regulated Expression of HuEPO in C3H/H and Balb/c Mice

The presence of HuEPO in the plasma of treated C3H/H mice was assessedby ELISA. HuEPO expression was detected with both AAV-huEPO vectors. Asshown in FIG. 9, HuEPO expression levels were four times higher than thenormal human physiological levels (normal human EPO levels are 4 to 24mU/ml). Plasma HuEPO levels were ten times higher in animals treatedwith 0034A compared with animals that received the 0034B vector. NoHuEPO expression was detected in the control animals. HuEPO expressionlevels peaked between day 7 and 14 and remained steady from day 14 to28.

Importantly, HuEPO expression paralleled HCT increases and displayed anexpected lag time between the induction of huEPO expression andmeasurable HCT changes (time necessary for the red blood cells toproliferate in response to EPO). Ligand was removed on day 29. No HuEPOwas detected in the plasma at days 35, 42, and 50 (vector-treated andcontrol). At Day 50, the relevant animals were put back on chowcontaining activator ligand. HuEPO expression again was detectable atlevels similar to what was observed in the first induction cycle. Ligandwas removed on day 64. HuEPO expression was not detectable on day 71,and was not detectable with the 0034A vector at day 85. Low levels ofHuEPO expression were detected with the 0034B vector at day 85.

An identical study to that displayed in FIG. 9 was performed in parallelusing normal Balb/c mice (FIG. 10).

Example 6 Regulated Expression of HuEPO Following a Single IM Injectionof AAV-HuEPO

Animals were dosed via IM injection of 10¹¹ vp of AAV-HuEPO (0034B) onday 0. Ligand was delivered on a daily basis during the first 21 days ofthe study via oral gavage (OG). The presence of HuEPO in the plasma ofthe treated mice was assessed by ELISA and HuEPO expression paralleledHCT changes. As shown in FIG. 11, HCT levels and HuEPO expression peakedon day 21. Ligand delivery was stopped on day 21 and huEPO expressionand HCT displayed a sharp decrease when activator ligand was notpresent. HuEPO ELISA data for the day 35 samples were not yet availablewhen FIG. 11 was prepared.

HCTs were compared to the baseline (time 0, pre-bleed) for each animaland group averaged for each time point. As shown in FIGS. 48A and 48B,an up to 1.7 time increase in HCT at 14 days with 0034A in C3H/H miceand 28 days in Balb/c mice. HCT levels increased up to 1.4 timesfollowing administration with 0034B in C3H/H and up to 1.6 times inBalb/c mice. HCT decreased to within the normal range following ligandinducer removal.

Example 7 Regulated Expression of HuEPO by Activator Ligand Dose

Animals were bled for baseline HCT levels, followed by IM administrationof 10¹¹ AAV-HuEPO viral genomes per animal (0034A). Animals receivedactivator ligand-containing chow, with activator ligand concentrations1000 mg/kg (18-1000), 250 mg/kg (18-250), 100 mg/kg (18-100), 50 mg/kg(18-50), or 0 mg/kg (18-0) starting on the day of AAV administration.Control animals received either vector administration and normal chow(no activator ligand, 18-0), or they received vehicle-alone IMadministration (saline) with (18-1000) or without (18-0) activatorligand. HCT and HuEPO expression levels were measured in all groupsevery 7 days.

As shown in FIG. 13, no HuEPO was detected in the baseline, prebleedsamples. HuEPO expression was detected on day 7 to 21. At the lowestconcentration of activator ligand (18-50) HuEPO expression was notdetected above the limit of sensitivity of the assay (8 mU/ml) at 7days, but was detectable at 14 and 21 days, albeit at lower levels thananimals that received the higher activator ligand doses. HuEPO reached asimilar peak expression level in cohorts that received the two highestconcentrations of activator ligand (18-1000 and 18-250). HuEPO levels inthe plasma of animals that were on the intermediate concentration ofactivator ligand (18-100) displayed lower HuEPO expression at 7 dayswhich reached to similar levels as the 18-1000 and 18-250 animals atdays 14 and 21. HCT changes paralleled HuEPO expression levels.Following ligand removal on day 21 HCT levels decreased to with thenormal range.

These data demonstrate regulated HuEPO expression following a singleintramuscular administration of AAV vector. Importantly, HuEPOexpression levels were regulated by activator ligand dose. These datasuggest that HuEPO expression may be maintained within the normalphysiological range by titration of the activator ligand concentration,and has clinical application.

Example 8 Regulated Expression of HuEPO Following a Single IM Injectionof AAV-HuEPO

A total of 25¾-nephrectomized C3H/H mice were obtained from Taconic.Animals were bled for baseline HuEPO levels and HCT. The indicatedcohorts received RG-115932 activator ligand in the chow (18-1000, 1000mg/kg chow) for the duration of the study. On day 0, RTS-HuEPO plasmidDNA (0034D) was administered through open muscle IM injection withelectroporation (EP) one hour following pretreatment with Hyase (50 UrHUPH20 from Halozyme). The animal cohorts included 1) HuEPODNA+EP+Hlyase+ligand, 2) HuEPO DNA+EP+Hyase without ligand, 3) HuEPODNA+EP, no Hyase, +ligand, 4) Saline+EP+Hyase, +ligand, and 5) animalstreated with IP injections of recombinant human EPO protein twice perweek.

As shown in FIG. 14, high HuEPO expression was detected in the plasma onday 8 and decreased to a lower normal range on day 22. HCT changesparalleled HuEPO levels in the plasma. HCT changes were not detected inthe plasma following re-administration of the vector on day 30.

HuEPO plasmid DNA (0034C, RTS-hEpo-IRES-fLuc) was delivered to Balb/cmice (quadriceps, 200 ug of DNA per animal), followed byelectroporation. Animals were also treated one hour prior to DNAdelivery and electroporation with hyaluronidase (50 U rHUPH20 fromHalozyme), or saline alone. All animals received activator ligand viachow for the duration of this study.

HCTs were measured in mice prior to DNA delivery and at Day 4 and Day 7after delivery. As shown in FIG. 15, a 40% increase in HCT at 7 days wasdetected in animals that received the huEPO plasmid plus hyaluronidasepretreatment, while animals that received huEPO without hyaluronidasepretreatment displayed a 25% increase in HCT at 7 days.

It is to be understood that the foregoing described embodiments andexemplifications are not intended to be limiting in any respect to thescope of the invention, and that the claims presented herein areintended to encompass all embodiments and exemplifications whether ornot explicitly presented herein.

LITERATURE

-   Abdalla, 2007.

1-6. (canceled)
 7. A vector comprising a polynucleotide encoding a geneswitch, wherein the polynucleotide comprises (1) at least onetranscription factor sequence which is operably linked to a promoter,wherein the at least one transcription factor sequence encodes aligand-dependent transcription factor, and (2) a polynucleotide encodingone or more proteins operably linked to a promoter which is activated bythe ligand-dependent transcription factor, wherein the one or moreproteins is selected from the group consisting of a C1 esteraseinhibitor, a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, aprostaglandin synthase, a glucagon-like peptide-1 (GLP-1), aglucagon-like peptide-2 (GLP-2), adiponectin, leptin, and cysticfibrosis transmembrane conductance regulator (CFTR).
 8. The vector ofclaim 7, wherein one or more of the proteins is a human protein.
 9. Thevector of claim 7, wherein the vector is a viral vector.
 10. The vectorof claim 9, wherein the viral vector is selected from the groupconsisting of an adenovirus, an adeno-associated virus, a retrovirus, apox virus, a baculovirus, a vaccinia virus, a herpes simplex virus, anEpstein-Barr virus, a geminivirus, a pseudorabies virus, a parvovirus,and a caulimovirus virus vector.
 11. The vector of claim 7, wherein thegene switch is an ecdysone receptor (EcR)-based gene switch.
 12. Thevector of claim 7, wherein the polynucleotide encoding a gene switchcomprises a first transcription factor sequence under the control of afirst promoter and a second transcription factor sequence under thecontrol of a second promoter, wherein a first transcription factorencoded by the first transcription factor sequence and a secondtranscription factor encoded by the second transcription factor sequenceinteract to form a complex which functions as a ligand-dependenttranscription factor.
 13. The vector of claim 7, wherein thepolynucleotide encoding a gene switch comprises a first transcriptionfactor sequence and a second transcription factor sequence under thecontrol of a promoter, wherein a first transcription factor encoded bythe first transcription factor sequence and a second transcriptionfactor encoded by the second transcription factor sequence interact toform a complex which functions as a ligand-dependent transcriptionfactor.
 14. The vector of claim 13, wherein the first transcriptionfactor sequence and the second transcription factor sequence areconnected by an EMCV internal ribosomal entry site (IRES).
 15. Thevector of claim 7, wherein one of the one or more proteins comprises anamino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequence encoded by SEQ ID NO: 9, 10,or 11; or wherein one of the one or more proteins comprises an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 12, 13, 14, 15, 17,18, 19, 20 or
 21. 16-22. (canceled)
 23. A method of producing apopulation of cells expressing one or more proteins, wherein the methodcomprises modifying the cells with a vector according to claim
 7. 24-30.(canceled)
 31. A population of cells produced according to the method ofclaim
 23. 32-69. (canceled)
 70. A composition comprising the vector ofclaim 7, or the population of cells of claim 31, and a pharmaceuticallyacceptable carrier.
 71. The composition of claim 70, which isadministered systemically, intravenously, intratumorally, orally,intraperitoneally, intramuscularly, intravertebrally, intracerdbrally,intrathecally, intradermally, or subcutaneously. 72-73. (canceled)
 74. Akit comprising the vector of claim 7, or the population of cells ofclaim
 31. 75. (canceled)
 76. The vector of claim 7, wherein the ligandthat activates the ligand-dependent transcription factor is adiacylhydrazine.
 77. The vector of claim 76, wherein the diacylhydrazineis RG-115819, RG-115830 or RG-115932.
 78. The vector of claim 7, whereinthe ligand that activates the ligand-dependent transcription factor isan amidoketone or oxadiazoline. 79-81. (canceled)
 82. A kit comprisingthe vector of claim 7, or the population of cells of claim 31, and aligand.
 83. The kit of claim 82, wherein the ligand is adiacylhydrazine.
 84. The kit of claim 83, wherein the diacylhydrazine isRG-115819, RG-115830 or RG-115932.
 85. The kit and ligand of claim 82,wherein the ligand is an amidoketone or oxadiazoline.